Laurová, T. Application of model CAPM on European stock market. Diploma thesis. Brno: Mendel University, 2015. The fundamental of this thesis is application of CAPM model on European stock market. Diploma thesis consists of two parts. The first one is the theoretical part, which is describing the basics of CAPM model, which serves for better understanding of the model and portfolio management. In the second, practical part, there will be solved the basic characteristics of model. There will be also created portfolios in Microsoft Excel. Finally there will be evaluated the veracity of the specified hypothesis and comparison of incurred portfolios.

The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate

Class of 2016 Abstract / Objectives: Patient medication adherence is a basic requirement for treating chronic myelogenous leukemia (CML) with oral tyrosine kinase inhibitors (TKIs). When imatinib adherence rates are less than 80 or 90 percent, major and complete molecular responses, respectively, do not happen. The purpose of this study was to determine the effect of a real-time medication monitoring (RTMM) reminder system adherence program on the medication possession ratio (MPR). Methods: This analytic study was a retrospective cohort study and used data extracted from chart reviews for patients who received services from 2011 to 2015. It was approved by the Institutional Review Board. The study consisted of an intervention group and a control group (50 patients each). MPRs, demographic, descriptive, and categorical variables were summarized using means, standard deviations (SD), and frequencies/percentages. Results: The study population consisted of adult patients (mean age=62.2, SD=2.7, 50% male) treated by Avella Specialty Pharmacy who received imatinib or nilotinib as treatment for CML, gastrointestinal stromal tumors (GIST), or a similar positive Philadelphia chromosome cancer. Only 4% of patients in the intervention group had an < 85% MPR, compared to 46% in the control group (p < 0.001). Conclusions: In those patients who had an MPR of ≥ 85%, the difference between the groups was statistically significant. As past studies have shown, adherence rates greater than 90% have a higher likelihood of a major or complete molecular response and a greatly reduced risk of disease progression.

No description available.

The presence of the translocation t(9;22)(q34;q11), leading to the BCR::ABL1 fusion transcript, is the hallmark of chronic myeloid leukemia (CML). Nevertheless, atypical presentation at diagnosis can be challenging. However, although most patients with CML are diagnosed with the e13a2 or e14a2 BCR:: ABL1 fusion transcripts, about 5% of them carry rare BCR::ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3, and e6a2. In particular, the e6a2 fusion transcript has been associated with clinically aggressive disease frequently presenting in accelerated or blast crisis phases. To date, there is limited evidence on the efficacy of front-line second-generation tyrosine kinase inhibitors for this genotype. Here, we report two patients, in whom the diagnosis of CML was challenging. The use of primers recognizing more distant exons from the common BCR::ABL1 breakpoint region correctly identified the atypical BCR::ABL1 e6a2 fusion transcript. Treatment with the second-generation tyrosine kinase inhibitor nilotinib was effective in our patient expressing the atypical e6a2 BCR::ABL1 fusion transcript.

Recruitment of mural cells, i.e. pericytes and smooth muscle cells (SMC), is essential to improve the maturation of newly formed vessels. One of the major factors involved in this process is the endothelial cell-secreted Platelet-Derived Growth Factor BB (PDGF BB). Sonic hedgehog (Shh) has also been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms involved have not yet been elucidated. We first identified Shh as a target of PDGF BB and found that SMC respond to Shh not only by upregulating the Gli1-dependent canonical pathway, but also by activating ERK1/2 and PI3K-dependent non-canonical pathways. Moreover, we found that PDGF BB-induced SMC migration, involves Shh-dependent PI3K, ERK1/2 and Gli1 activation. In the mouse model of corneal angiogenesis, PDGF BB and Shh were expressed by endothelial cells and mural cells of VEGF-induced newly formed blood vessels, respectively. PDGF BB inhibition reduced Shh expression, confirming that Shh is a target of PDGF BB, as demonstrated by in vitro experiments. Finally, we found that inhibition of either PDGF BB or Shh signaling reduced NG2+ mural cell recruitment into neovessels and subsequently reduced the neo-vessel lifespan. In this work, we demonstrate, for the first time, that Shh is a key mediator of PDGF BB-induced mural cell migration and recruitment into neo-vessels and elucidates the molecular signaling pathway involved in this process. / Recruitment of mural cells, i.e. pericytes and smooth muscle cells (SMC), is essential to improve the maturation of newly formed vessels. One of the major factors involved in this process is the endothelial cell-secreted Platelet-Derived Growth Factor BB (PDGF BB). Sonic hedgehog (Shh) has also been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms involved have not yet been elucidated. We first identified Shh as a target of PDGF BB and found that SMC respond to Shh not only by upregulating the Gli1-dependent canonical pathway, but also by activating ERK1/2 and PI3K-dependent non-canonical pathways. Moreover, we found that PDGF BB-induced SMC migration, involves Shh-dependent PI3K, ERK1/2 and Gli1 activation. In the mouse model of corneal angiogenesis, PDGF BB and Shh were expressed by endothelial cells and mural cells of VEGF-induced newly formed blood vessels, respectively. PDGF BB inhibition reduced Shh expression, confirming that Shh is a target of PDGF BB, as demonstrated by in vitro experiments. Finally, we found that inhibition of either PDGF BB or Shh signaling reduced NG2+ mural cell recruitment into neovessels and subsequently reduced the neo-vessel lifespan. In this work, we demonstrate, for the first time, that Shh is a key mediator of PDGF BB-induced mural cell migration and recruitment into neo-vessels and elucidates the molecular signaling pathway involved in this process.

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.

Les cellules musculaires lisses (CML) vasculaires les composants cellulaires principaux de la paroi artérielle, sont exposées constamment aux contraintes mécaniques. Les contraintes mécaniques cycliques régulent de nombreuses fonctions des CML vasculaires via les intégrines. Parmi les intégrines, l'[alpha]v[gamma]3 est non seulement un mécano-transducteur mais aussi le récepteur de la prothrombine à la surface des CML. L?activation de l'intégrine [alpha]v[gamma]3 par les contraintes mécaniques pourrait favoriser l'adhésion des CML à la prothrombine et aussi accélérer la génération de thrombine à la surface des CML. Pour vérifier cette hypothèse, nous avons étudié l'effet des contraintes mécaniques sur la génération de thrombine par les CML et identifié les voies de la signalisation impliquées. Nous avons utilisé un modèle de Flexcell utilisant les CML aortiques de rat, soumises à un étirement cyclique (10%, 1Hz). L'exposition à l'étirement cyclique pendant 1h et 6h induit un phénotype de différenciation et non-apoptotique des CML et une augmentation de l'expression de l'intégrine [alpha]v[gamma]3. Il y a aussi une augmentation de la phosphorylation de Src, FAK, AKT de façon temps dépendant et une augmentation de la phosphorylation de l'ILK à 15 min et du clivage de taline de 5 à 60 min. L'étirement cyclique augmente l'adhésion des CML à la prothrombine et la génération de thrombine avec un effet maximum à 6h de 67% et 30% respectivement. Le peptide mimétique de l'intégrine [alpha]v[gamma]3 (cRGDPV) et le siARN [alpha]v bloquent tous les effets de l'étirement cyclique sur les CML. Le siARN taline inhibe l'expression de la sous-unité [alpha]v et également la phosphorylation de Src, AKT et ILK. Le siARN ILK n'a pas d'effet sur l'expression de l'[alpha]v mais inhibe la phosphorylation d'AKT et le clivage de taline à 6h de l'étirement cyclique. Ainsi, l'étirement cyclique induit une plus forte génération de thrombine par les CML vasculaires via l'activation des voies de signalisation dépendante de l'[alpha]v[gamma]3. Cette étude suggère que la génération de thrombine intravasculaire peut être régulée par des antagonistes de l'intégrine [alpha]v[gamma]3 et peut devenir une nouvelle cible thérapeutique chez les patients avec une pression pulsée élevée / Vascular smooth muscle cells (SMC), the main cellular components of the arterial wall, are constantly exposed to mechanical stretch. Cyclic mechanical stress regulates many functions of vascular SMC via integrins. Among the integrins, [alpha]v[gamma]3 is not only a mechanotransducer but also the receptor of prothrombin in the vascular SMC. Activation of integrin [alpha]v[gamma]3 by mechanical stretch may promote SMC adhesion to prothrombin and also accelerate thrombin generation on the surface of SMC. To test this hypothesis, we have studied the effect of mechanical stretch on the generation of thrombin by SMC and identified possible signaling pathway involved. We used a Flexcell model using rat aortic SMC subjected to cyclic stretch (10%, 1Hz). Exposure to cyclic stretch for 1h and 6h induced a phenotype of differentiation and non-apoptosis of SMC and an increased expression of integrin [alpha]v[gamma]3. There was also an increase in phosphorylation of Src, FAK, and AKT in a time dependent manner, increased phosphorylation of ILK at 15min and the cleavage of talin from 5 to 60min. Cyclic stretch increased the adhesion of prothrombin to the SMC, and thrombin generation with a maximum effect of 67% and 30% respectively. A peptide mimetic of integrin [alpha]v[gamma]3 (cRGDPV) and [alpha]v siRNA both blocked all the effects of cyclic stretch on SMC. A talin siRNA inhibited the expression of [alpha]v and the phosphorylation of Src, AKT and ILK. An ILK siRNA has no effect on the expression of [alpha]v but inhibited the phosphorylation of AKT and the cleavage of talin at 6h of stretch. Thus, cyclic stretch induced a higher thrombin generation by vascular SMC via activation of signaling pathways dependant on [alpha]v[gamma]3. This study suggests that intravascular thrombin generation can be regulated by antagonists of integrin [alpha]v[gamma]3 and can become a new therapeutic target for the patients with a high pulse pressure

Chronic Myelogenous Leukemia (CML) is a blood cancer that arises when hematopoietic cells acquire an abnormal protein known as BCR-ABL. Current therapies for CML include drugs that inhibit BCR-ABL. However, these drugs only suppress the disease and do not cure it. One reason is that BCR-ABL drugs fail to kill the primitive population of CML cells, referred to as leukemia stem cells (LSCs), which are responsible for initiating and propagating CML. Since LSCs are not killed, the cancer is not cured and many affected patients eventually relapse. Recent studies suggest that LSCs are protected from current therapies by the bone marrow micro-environment where they reside. There, cytokine signaling molecules are present, which mediate processes that protect LSCs from BCR-ABL drugs. The stem cell factor (SCF) is one of these signaling molecules. It activates the receptor c-Kit located on the surface of LSCs, and this activation in turn allows proliferating LSCs to resist BCR-ABL drugs, even without prior exposure to these drugs, i.e., innate resistance is observed. In this thesis, the mechanism of this innate resistance is investigated, so that a suitable treatment strategy can be developed. To this end, a co-agent approach based on synthetic antibodies (sABs) is proposed to inhibit the receptor c-Kit, with the goal of disrupting its activation by the ligand SCF. This disruption should in turn block the SCF-mediated innate resistance, thus potentially restoring BCR-ABL drug apoptotic activity. The method for this disruption involves targeting the c-Kit structural susceptibility. Specifically, the sABs are designed via antibody phage display technology to target the D1–D2–D3 domains representing the SCF binding sites, hence preventing downstream pathway activation. The hypothesis is that, by blocking the SCF-mediated innate resistance, a suitable combination of such an sAB co-agent and a BCR-ABL drug should be conducive to suppressing LSCs, thereby providing a potential means to improve CML treatment. In addition, to assess the performance of the proposed treatment strategy, a set of in vitro tests is conducted, focusing on performance behaviors such as cell binding, cell death, and the progenitor inhibition. The experimental results support the hypothesis that the proposed combinatorial strategy is indeed a promising approach to mitigate the innate resistance, thus restoring BCR-ABL drug apoptotic activity.

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.