Abstract In our dissertation we address the needs rooting in the ongoing process of systematic efforts to improve the Czech educational system. Mainly, we focus on the question of how to implement the cross-curricular topic Multicultural Education into English language classes at lower and upper secondary schools. As Multicultural Education, along with the other cross-curricular topics, has become an obligatory and essential part of the reformed Czech curricula. The author of this dissertation is employed with the English Department at the Faculty of Education of The University of South Bohemia in České Budějovice. That is why we aim to introduce the theoretical background and history of the cross-curricular topic Multicultural Education in the Czech educational system. Then, based on that theoretical background, we shall design and run a course at the Pedagogical faculty for our students (teachers-to-be) that will provide them with sufficient amount of theory. Within the course the students will be given enough practical examples of the implementation of Multicultural Education into English classes when combining cultural and linguistic aims and at the end of the course we will allow the students to design and try such a class during their peer teaching. We will also publish the results of the course for...

JAK/STAT signaling pathway is one of the most studied intracellular cascades transmitting signals from the extracellular environment to the cell nucleus in order to affect expression of target genes. Circadian clocks localized in the suprachiasmatic nuclei (SCN) of the hypothalamus are sensitive especially to light but they can respond to non-photic stimuli such as growth factors, opioids, leptin and cytokines that have been demonstrated to perform its function via the JAK/STAT signaling pathway. The recent findings of our laboratory demonstrated that STAT3 protein is highly produced by SCN of rat. Primary aim of our experiments was to test the circadian regulation of STAT3 production in SCN and describe the effect of exogenously administered leptin on STAT3 phosphorylation in the SCN, pineal gland and hypothalamic structures responsible for regulated feeding behavior and energy metabolism. Because activation of leptin receptors may stimulate a number of other signaling cascades, we chose phosphorylated forms of kinase ERK1/2 and GSK-3β as other markers of intracellular changes after administration of leptin in the studied structures. Our results proved rhythmic production of STAT3 protein in SCN of rat and indicated circadian regulation of sensitivity to leptin in hypothalamic structures. The data...

This diploma thesis The Specifics of Internet Broadcasting in the Czech Republic with Focus on the Viewers' Comments on the Show Jak nás vidí svět (Stream.cz) deals with the typical features of online television broadcasting in the Czech Republic and their comparison to the typical features of traditional television broadcasting. In the practical part, the thesis focuses on the viewers' comments on the show Jak nás vidí svět. The main aim of the thesis is to describe the current position of internet television broadcasting in the Czech Republic with its specifics and its differences compared to traditional broadcasting, focusing on the show Jak nás vidí svět (Stream.cz), specifically on the viewers' comments on the show. First, traditional and internet broadcasting are defined and their specifics are analyzed in detail, which are then compared in a separate chapter. The aim of the first part of the thesis is to find and define the specifics of internet television broadcasting in the Czech Republic, especially in comparison with traditional television broadcasting, with regard to the ongoing media convergence and the fact that the individual forms converge and sometimes intersect. The practical part is based on one of the typical features of online broadcasting, namely participation in the form of...

Οι όγκοι εγκεφάλου του ανθρώπου είναι φαινοτυπικά και γονοτυπικά ετερογενείς. Υπάρχουν σημαντικά κενά στην κατανόηση των μοριακών οδών που εμπλέκονται στην γένεση και ανάπτυξη των νεοπλασμάτων αυτών, καθώς και στη βιολογική και κλινική συμπεριφορά τους. Δεδομένου ότι: α) υπάρχει ένας αυξανόμενος όγκος πληροφοριών σχετικά με την εμπλοκή των υποδοχέων νευροτροφινών Trks (υποδοχείς κινάσης τυροσίνης) στην παθογένεια των νεοπλασμάτων του ΚΝΣ, β) οι νευροτροφίνες μπορούν να ρυθμίζουν τη γονιδιακή έκφραση στο νευρικό σύστημα μέσω του μεταγραφικού παράγοντα STAT-5 με μηχανισμό ανεξάρτητο της ενεργοποίησης της κινάσης JAK-2, γ) η οδός μεταγωγής σήματος JAK-2/STAT-5 ενεργοποιείται από την ερυθροποιητίνη (Epo) (μέσω δέσμευσής της με τον υποδοχέα της EpoR) η οποία πρόσφατα έχει θεωρηθεί ως παράγοντας-κλειδί της αύξησης των όγκων λόγω των αντιαποπτωτικών και αγγειογενετικών ιδιοτήτων και δυνητικός στόχος ογκολογικών θεραπευτικών στρατηγικών, δ) η Epo διαντιδρά με τον παράγοντα αγγειογένεσης VEGF ενδυναμώνοντας την αγγειοδραστικότητά του και συχνά οι δύο αυτοί αναπτυξιακοί παράγοντες συνεκφράζονται, ε) ερευνητικά δεδομένα υποδεικνύουν ένα δυνητικό ρόλο του άξονα VEGF/JAK-2/ STAT-5 στην αγγειογένεση στον καρκίνο και στ) σε πολλά νεοπλάσματα, τα κύτταρα του όγκου καθώς και τα μακροφάγα του στρώματος εκφράζουν τους παράγοντες λεμφαγγειογένεσης VEGF-C και VEGF-D οι οποίοι μέσω του υποδοχέα τους VEGFR-3 (υποδοχέας κινάσης τυροσίνης) έχει βρεθεί ότι προάγουν την αγγειογένεση στα νεοπλάσματα αυτά, ο στόχος της παρούσας εργασίας είναι η μορφολογική μελέτη της έκφρασης των υποδοχέων κινάσης τυροσίνης Trks (υποδοχείς νευροτροφινών) και VEGFR-3 και η συσχέτισή τους με την οδό μεταγωγής σήματος EpoR/ JAK-2/ STAT-5 στα νεοπλάσματα του εγκεφάλου στον άνθρωπο. Για την ανίχνευση των Trks, EpoR, JAK-2, STAT-5 και VEGFR-3, χρησιμοποιήθηκε η ανοσοϊστοχημική μέθοδος σε διαδοχικές τομές ιστού πάχους 4μm, μονιμοποιημένου σε φορμόλη και εγκλεισμένου σε παραφίνη. Χρησιμοποιήθηκαν ειδικά αντισώματα έναντι των προς μελέτη πρωτεϊνών. Εξετάσθηκαν συνολικά 92 όγκοι εγκεφάλου (50 αστροκυτώματα: 5 πιλοκυτταρικά αστροκυττώματα WHO grade I, 6 διάχυτα αστροκυττώματα WHO grade II, 10 αναπλαστικά αστροκυττώματα. WHO grade III και 29 πολύμορφα γλοιοβλαστώματα. WHO grade IV, 8 ολιγοδενδρογλοιώματα, 6 επενδυμώματα, 5 μυελοβλαστώματα και 23 μηνιγγιώματα). Επιπλέον, στη μελέτη συμπεριλήφθηκαν τομές ιστού φυσιολογικού εγκεφάλου. Στην παρούσα εργασία, η πλειονότητα των αστροκυττωμάτων ανεξαρτήτως βαθμού κακοήθειας, έδειξε μέτρια έως έντονη κυτταροπλασματική ανοσοδραστικότητα για τους υποδοχείς TrkA (57%), TrkB (56%) και TrkC (46%). Όλα τα ολιγοδενδρογλοιώματα και τα επενδυμώματα που εξετάσθηκαν ήταν αρνητικά για τους υποδοχείς Trk ενώ έκφραση των υποδοχέων ανιχνεύθηκε σε μικρό ποσοστό των μυελοβλαστωμάτων. Στις ανοσοθετικές περιοχές των όγκων, το τοίχωμα των αγγείων ήταν έντονα θετικό για τον υποδοχέα TrkB. Σε ένα σημαντικό ποσοστό αστροκυττωμάτων (41%), επενδυμωμάτων (33%) και μηνιγγιωμάτων (48%) ανιχνεύθηκε έκφραση του EpoR, ενώ τα ολιγοδενδρογλοιώματα και τα μυελοβλαστώματα που εξετάσθηκαν ήταν αρνητικά. Συνέκφραση των JAK-2/STAT-5 ανιχνεύθηκε στο 39% των αστροκυττωμάτων, στο 43% των ολιγοδενδρογλοιωμάτων, στο 50% των επενδυμωμάτων και σε όλα (100%) τα μυελοβλαστώματα που συμπεριλήφθηκαν στη μελέτη. Η πλειονότητα των μηνιγγιωμάτων έδειξε ασθενή έως μηδενική έκφραση των πρωτεινών JAK-2 και STAT-5. Σε κάποιους όγκους, ανιχνεύθηκε έκφραση της πρωτείνης STAT-5 ενώ ήταν αρνητικοί για την κινάση JAK-2. Επίσης, βρέθηκαν όγκοι JAK-2- ανοσοθετικοί/ EpoR-ανοσοαρνητικοί. Στα ενδοθηλιακά κύτταρα των αγγείων των όγκων ανιχνεύθηκε έκφραση των EpoR, JAK-2 και/ή STAT-5. Η μελέτη της έκφρασης του VEGFR-3 έδειξε την καθολική σχεδόν έκφραση του υποδοχέα στο ενδοθήλιο των αγγείων, στους όγκους εγκεφάλου που εξετάσθηκαν, καθώς και στο τοίχωμα μεγαλύτερων αγγείων εκτός της περιοχής των όγκων. Επίσης, σε κάποιες περιπτώσεις, παρατηρήθηκε έκφραση του υποδοχέα σε νεοπλασματικά κύτταρα. Στα αστροκυτταρικά γλοιώματα, η έκφραση των pan-Trk, TrkB και TrkC σχετίζεται στατιστικά σημαντικά με την έκφραση STAT-5 ενώ δεν υπάρχει στατιστικά σημαντική συσχέτιση της έκφρασης του υποδοχέα TrkA με την έκφραση των JAK-2 και STAT-5, καθώς και της έκφρασης των Trks με την έκφραση της κινάσης JAK-2. Στα αστροκυτταρικά γλοιώματα, η έκφραση JAK-2 σχετίζεται στατιστικά σημαντικά με την έκφραση STAT-5, ενώ η έκφραση των JAK-2 και STAT-5 δεν σχετίζεται σημαντικά με την έκφραση EpoR. Στα επενδυμώματα υπάρχει στατιστικά σημαντική συσχέτιση μεταξύ της έκφρασης JAK-2, STAT-5 και EpoR. Επίσης, στα μυελοβλαστώματα υπάρχει στατιστικά σημαντική συσχέτιση μεταξύ της έκφρασης JAK-2 και STAT-5. Σε σημαντικό ποσοστό των εξετασθέντων όγκων –ιδιαίτερα των γλοιωμάτων- συνεκφράζονται οι υποδοχείς EpoR και VEGFR-3 στο ενδοθήλιο και στα λεία μυικά κύτταρα των αγγείων. Συγκεκριμένα, έκφραση και των δύο υποδοχέων στο τοίχωμα των αγγείων του όγκου, ανιχνεύθηκε, στο 58% των αστροκυτταρικών γλοιωμάτων, στο 67% των ολιγοδενδρογλοιωμάτων, στο 50% των επενδυμωμάτων, στο 20% των μυελοβλαστωμάτων και στο 33% των μηνιγγιωμάτων. Επίσης συνέκφραση των υποδοχέων TrkB και VEGFR-3 στο ενδοθήλιο των αγγείων των όγκων ανιχνεύθηκε στο 57% των αστροκυτταρικών γλοιωμάτων. Η έκφραση στο ενδοθήλιο των αγγείων των όγκων (α) του υποδοχέα TrkB στην πλειονότητα των TrkB-ανοσοθετικών όγκων, (β) των EpoR, JAK-2 και/ή STAT-5 σε αρκετούς όγκους ανεξάρτητα της ανοσοθετικότητας των νεοπλασματικών κυττάρων για τις πρωτεΐνες αυτές και (γ) του VEGFR-3 σχεδόν σε όλους του εξετασθέντες όγκους, οδηγεί στο συμπέρασμα ότι οι παραπάνω παράγοντες πιθανόν συνεργάζονται στην αγγειογενετική διαδικασία στους όγκους εγκεφάλου. Η παρούσα μελέτη απέδειξε την παρουσία των: Trks, EpoR, JAK-2, STAT-5 και VEGFR-3 σε σημαντικό ποσοστό των όγκων εγκεφάλου στον άνθρωπο. Είναι απαραίτητο να διενεργηθούν λειτουργικές μελέτες των σηματοδοτικών οδών που προκύπτουν από την ενεργοποίηση των Trks, EpoR και VEGFR-3, προκειμένου να αποσαφηνισθούν οι πιθανές διαντιδράσεις αυτών των οδών στη διαδικασία της ογκογένεσης, γεγονός που θα μπορούσε να οδηγήσει στο σχεδιασμό νέων θεραπευτικών στρατηγικών για την αποτελεσματική αντιμετώπιση των όγκων εγκεφάλου στον άνθρωπο. / Brain tumors are phenotypically and genotypically heterogeneous. Significant gaps exist in current understanding of the molecular pathways involved in the genesis, progression, and biological and clinical behavior of brain tumors. Considering that : a) there is a growing amount of evidence implicating the neurotophin receptors Trks (receptor tyrosine kinases) in the pathogenesis of CNS tumors, b) neurotrophins may regulate neuronal gene expression via transcription factor STAT-5 in a JAK-2 independent manner, c) the JAK-2/STAT-5 signaling pathway is activated by Epo (recently characterized as a key factor for tumor growth) through its receptor EpoR, d) Epo is known to interact synergistically with vascular endothelial growth factor (VEGF), a potent angiogenic factor, and enhance its vascular activity, and these two growth factors, are often co-expressed, e) recent data suggest a potential role for a VEGF/JAK-2/STAT-5 axis in tumor angiogenesis and f) in many tumors, VEGF-C and -D are produced by tumor cells as well as by tumor-associated macrophages and through their receptor VEGFR-3 are involved in tumor angiogenesis and growth, the aim of this thesis is the morphological study of the expression patterns of the receptor tyrosine kinases Trks (neurotrophin receptors) and VEGFR-3 and their correlation with the EpoR/ JAK-2/ STAT-5 signaling pathway in human brain tumors. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded, 4μm thick serial sections using the anti-Mouse/ Rabbit Poly HRP IHC Detection kit (CHEMICON International, Inc.). Antibodies against the Trk receptors, EpoR, JAK-2, STAT-5 and VEGFR-3, were used. A total of 92 brain tumors (50 astrocytic gliomas: 5 pilocytic astrocytomas; WHO grade I, 6 diffuse fibrillary astrocytomas; WHO grade II, 10 anaplastic astrocytomas; WHO grade III and 29 glioblastomas multiforme; WHO grade IV, 8 oligodendrogliomas, 6 ependymomas, 5 medulloblastomas, and 23 meningiomas) were included in this study. Normal human brain tissue was obtained postmortem (2 males). The present study demonstrated moderate to strong, granular cytoplasmic immunoreactivity in the majority of astrocytomas, for TrkA (57%), TrkB (56%) and TrkC receptors (46%), independently of grade. All the oligodendrogliomas and the ependymomas examined, were Trk immunonegative while a small percentage of medulloblastomas exhibited Trk immunopositivity. The endothelium of tumor vessels, in the immunopositive areas of the tumors, showed conspicuous immunoreactivity for TrkB receptor. A significant percentage of astrocytomas (41%), ependymomas (33%) and meningiomas (48%) displayed EpoR immunoreactivity. Oligodendrogliomas and medulloblastomas were EpoR- immunonegative. JAK-2/STAT-5 co-expression was detected in 39% of astrocytomas, 43% of oligodendrogliomas, 50% of ependymomas and in all (100%) the medulloblastomas examined. In contrast, most of the meningiomas showed weak or no immunoreactivity for JAK-2 and STAT-5 proteins. Some tumors exhibited STAT-5 immunoreactivity being JAK-2-immunonegative and others were JAK-2-immunopositive being EpoR- immunonegative. In some tumors - independently of EpoR immunoreactivity in tumors cells- the endothelium of tumor capillaries as well as florid angioproliferative changes and/or proliferations of smooth muscle cells showed conspicuous immunoreactivity for EpoR. Furthermore, endothelial cells of some tumor vessels showed JAK-2 and/or STAT-5 immunoreactivity. The present study demonstrated the almost catholic VEGFR-3 expression in the endothelium of tumor vessels in the brain tumor specimens examined, as well as in the smooth muscle cells in peritumoral vessels. In some tumors, the tumor cells expressed VEGFR-3receptor. In astrocytic gliomas, pan-Trk, TrkB and TrkC expression was significantly correlated with STAT-5 expression but not with JAK-2 whereas TrkA expression was not significantly correlated with either JAK-2 or STAT-5 expression. In astrocytic gliomas, JAK-2 expression was significantly correlated with STAT-5 expression whereas JAK-2 and STAT-5 expression were not significantly correlated with EpoR expression. In ependymomas, strong relationship between JAK-2, STAT-5 and EpoR expression was confined. Furthermore, in medulloblastomas, strong relationship between JAK-2 and STAT-5 expression was detected. A significant percentage of astrocytomas (58%), oligodendrogliomas (67%), ependymomas (50%), medulloblastomas (20%) and meningiomas (33%), displayed EpoR and VEGFR-3 co-expression in tumor vessels. Additionally, TrkB and VEGFR-3 co-expression was detected in the endothelium of tumor capillaries in 57% of the astrocytomas examined. The above results indicate the existence of a ligand (other than Epo)-dependent or independent JAK-2 activation that leads to the constitutive activation of STAT-5 as well as the existence of at least one mechanism, other than the Trk - dependent gene induction by STAT-5 that results to the latter’s constitutive activation in these tumors. Furthermore, the finding of EpoR, JAK-2 and/or STAT-5 immunoreactivity in endothelial cells of tumor vessels, supports the ongoing notion of an angiogenic role of Epo in tumor neovascularization. The presence of TrkB, EpoR, JAK-2, STAT-5 and VEGFR-3 in the endothelium of tumor vessels in the brain tumors specimens included in this study, implicate these factors as possible critical players in the process of angiogenesis. The present study demonstrates the expression of Trks, EpoR, JAK-2, STAT-5 and VEGFR-3 in a significant percentage of human brain tumors. Functional studies of the Trks, EpoR and VEGFR-3-activated signaling pathways, are necessary in order to clarify the meaning of a possible cross-talking between these pathways in the process of oncogenesis, the deep understanding of which, could lead to innovative new strategies for drug targeting to human brain tumors.

No description available.

The Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the t(9:22) genetic translocation and expression of the oncogenic tyrosine kinase BCR-ABL . A first BCR-ABL Tyrosine Kinase Inhibitor (TKI), Imatinib (IM), was identified that inhibits proliferation of BCR-ABL expressing hematopoietic cells and leads to disease remission. However, BCR-ABL mRNA remains detectable in the most immature HSCs and discontinuation of IM results in clinical relapse. STAT5 factors play a crucial role in the CML pathogenesis of human primary CML cells. However, the contribution of the two related STAT5 genes, STAT5A and STAT5B, was unknown. We used an RNAinterference based strategy to analyze STAT5A or STAT5B roles in normal and CML cells. We showed that STAT5A/5B double knock-down (KD) triggers normal and CML cell apoptosis and suppressed long-term clonogenic potential of immature hematopoietic stem and progenitor cells known to be resistant to TKI treatment and responsible for residual disease. STAT5A loss alone was ineffective at impairing growth of both normal and CML cells under standard conditions. In contrast, STAT5A loss was sufficient to enhance Reactive Oxygen Species (ROS) which correlated with enhanced DNA damages in both normal and leukemic cells. We reported that STAT5A regulates oxidative stress through unconventional mechanisms, in a non-transcriptional-dependent manner. We further showed that, in contrast to primary cells at diagnosis, IM-resistant cells exhibited enhanced STAT5A dependence, by being sensitive to STAT5A single KD. To investigate the molecular basis of STAT5A activity in TKI-resistance and oxidative stress, we performed a transcriptomic analysis of STAT5 regulated genes. We identified Axl, which encodes a receptor tyrosine kinase, recently shown to be crucial in TKI-resistant CML cells. Specifically, Axl expression is enhanced by STAT5A. We investigated the role of Axl and we found that Axl KD did not affect survival of IM-sensitive CML cells. However, Axl KD decreased survival of IM-resistant cells, miming the activity of STAT5A. Moreover, Axl loss increased ROS levels in CML cells, promoting STAT5A anti-oxidant activity. We further sought to determine the expression of the Axl ligand, Gas6. Gas6 expression is dramatically reduced in CML primary cells at diagnosis compared to healthy cells. The strong and consistent down-regulation of Gas6 in CML cells suggested a possible role in the pathophysiology. Collectively, our findings highlight the pro-survival, stress protection and drug resistance roles of STAT5 factors, providing new understanding for medical treatment of CML patients. We suggest that STAT5A acts in synergy with Axl to face exogenous insults and propose a new mechanism by which CML cells increase their proliferation and reduce their motility by down-regulating Gas6 expression.

El curso clínico de la trombocitemia esencial (TE) se caracteriza por un elevado riesgo de aparición de complicaciones trombóticas, las cuales constituyen la principal causa de muerte en estos pacientes. Si bien la causa fundamental de la trombosis en esta enfermedad parece ser el aumento del número de plaquetas y sus alteraciones bioquímicas y funcionales, no existe una correlación clara entre estos factores y el desarrollo de trombosis. El objetivo de la presente tesis fue conocer el papel de la activación plaquetaria y leucocitaria en la trombosis de los pacientes con TE. Para ello, se determinó mediante citofluorometría el estado de activación plaquetaria y leucocitaria en 53 pacientes con TE (26 con historia de trombosis previa) y en otros tantos sujetos sanos de igual edad y sexo. Las determinaciones incluyeron la expresión de P-selectina en plaquetas (en condiciones basales y después del estímulo con ADP, trombina, ácido araquidónico [AA] o colágeno), los complejos plaqueta-neutrófilo y plaqueta-monocito, el CD11b en neutrófilos y monocitos y la expresión de factor tisular en los monocitos (FTm). Además, se investigaron el porcentaje de plaquetas reticuladas (PR), la resistencia adquirida a la proteína C activada (RAPCA), la expresión plaquetaria de factor tisular (FT) y las concentraciones plasmáticas de FT, factores de la coagulación, P-selectina soluble (sP-selectina), CD40 ligando soluble (sCD40L), antígeno del factor de von Willebrand (VWF:Ag), trombomodulina soluble (sTM), dímero D y fragmento 1+2 de la protrombina. Estos parámetros se correlacionaron también la presencia de la mutación V617F de JAK2 o su carga mutacional.En comparación con los controles, los pacientes con TE tenían una mayor expresión de P-selectina basal e inducida por trombina o AA, así como unos valores significativamente más elevados de complejos plaqueta-neutrófilo y plaqueta-monocito, expresión de CD11b en neutrófilos y expresión basal de FTm. La P-selectina basal e inducida por AA, el CD11b monocitario, la expresión de FTm inducida por lipopolisacárido, el porcentaje de PR, RAPCA y las concentraciones plasmáticas del factor V, factor VIII, VWF:Ag, sP-selectina y sCD40L se hallaron significativamente más elevadas en pacientes con historia de trombosis que en aquellos sin trombosis. En el estudio multivariado, el porcentaje de PR, la concentración de factor V y la presencia de RAPCA se asociaron independientemente con un aumento del riesgo de trombosis. Los pacientes con la mutación V617F de JAK2 tuvieron unos valores más elevados de P-selectina basal e inducida por AA que los pacientes sin esta condición. Finalmente, se objetivó un efecto dosis del porcentaje mutacional (>/- 12%), asociándose éste a una mayor concentración de FT, sP-selectina, sCD40L, FVW:Ag y menor de proteína S (PS).Estos resultados sugieren que la activación plaquetaria y monocitaria, así como la renovación plaquetaria, factor V y RAPCA pueden desempeñar un papel importante en la patogenia de la trombosis de los pacientes con TE, así como la posibilidad de la detección de pacientes con un mayor riesgo trombótico. La asociación entre la carga alélica de V617F con la disminución de PS o incremento de FT y de los marcadores solubles de activación plaquetaria y endotelial sugiere que dicha mutación podría promover un estado pretrombótico en la TE. EXTRACTO. Si bien la causa fundamental de la trombosis en la trombocitemia esencial (TE) parece ser el aumento del número de plaquetas y sus alteraciones bioquímicas y funcionales, no existe una correlación clara entre estos factores y el desarrollo de trombosis. Los resultados de la presente tesis doctoral sugieren que la activación plaquetaria y monocitaria, así como la renovación plaquetaria, factor V y la resistencia adquirida a la proteína C activada pueden desempeñar un papel importante en la patogenia de la trombosis de los pacientes con TE, así como la posibilidad de la detección de pacientes con un mayor riesgo trombótico. La asociación entre la carga alélica de V617F con la disminución de proteína S libre o incremento de factor tisular y de los marcadores solubles de activación plaquetaria y endotelial sugiere que dicha mutación podría promover un estado pretrombótico en la TE. PALABRAS CLAVE. Trombocitemia esencial, trombosis, activación plaquetaria, activación leucocitaria, coagulación, mutación V617F de JAK2. / "ROLE OF THE PLATELET AND LEUKOCYTE ACTIVATION IN THE PATHOGENESIS OF THE THROMBOSIS IN ESSENTIAL THROMBOCYTHEMIA", by EDUARDO ARELLANO RODRIGOTEXT:The mechanisms accounting for the increased risk of thrombosis in patients with essential thrombocythemia (ET) are not well known. The aim of the present thesis was to ascertain the role of platelet and leukocyte activation in the thrombosis of ET and its relationship with the JAK2 mutation.In 53 ET patients (26 with a history of thrombosis), platelet P-selectin (measured both at baseline and after stimulation with ADP, thrombin, arachidonic acid (AA), and collagen) and leukocyte CD11b expression, reticulated platelets (RP) percentage, platelet and monocyte tissue factor (mTF) expression, acquired activated protein C resistance (aAPCR), and plasma levels of tissue factor (TF), coagulation factors, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), thrombomodulin, D-dimer and prothrombin fragment 1+2 were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 mutational load. As compared with controls, ET patients had significantly higher values of baseline P-selectin and thrombin- and AA-induced platelet P-selectin expression, as well as higher platelet-neutrophil and platelet-monocyte complexes, neutrophil CD11b expression and baseline mTF expression. ET patients with thrombosis had significantly higher values for platelet P-selectin, monocyte CD11b, lipopolysaccharide-induced mTF expression and RP percentage, aAPCR, and levels of factors V and VIII, VWF:Ag, sP-selectin and sCD40L than patients without thrombosis and controls. At multivariate study, RP percentage, factor V levels, and aAPCR were independently associated with an increased risk of thrombosis. Patients with the JAK2 mutation showed higher baseline and AA-induced platelet P-selectin expression than those with wild-type allele. A mutant allele dosage effect ( 12 %) was observed for sP-selectin, sCD40L, TF, VWF:Ag and free protein S (PS) levels. These results support a role for platelet and monocyte activation, platelet turnover, factor V, and aAPCR in the thrombosis of ET, as well as their potential to identify patients at greater risk of thrombosis. The association between JAK2 V617F allele burden and either decreased free PS or increased TF and markers of platelet and endothelial activation could suggest that this mutation would promote an enhanced prethrombotic state in ET. EXTRACT. The mechanisms accounting for the increased risk of thrombosis in patients with essential thrombocythemia (ET) are not well known. The aim of the present thesis was to ascertain the role of platelet and leukocyte activation in the thrombosis of ET and its relationship with the JAK2 mutation. The results of the present thesis support a role for platelet and monocyte activation, platelet turnover, factor V, and aAPCR in the thrombosis of ET, as well as their potential to identify patients at greater risk of thrombosis. The association between JAK2 V617F allele burden and either decreased free PS or increased TF and markers of platelet and endothelial activation could suggest that this mutation would promote an enhanced prethrombotic state in ET. KEY WORDS. Essential thrombocythemia, thrombosis, platelet activation, leukocyte activation, coagulation, JAK2 V617F mutation.

[Truncated abstract] The innate ability of the heart to compensate for an increase in workload as a result of disease or injury, through an increase in size and mass is known as cardiac hypertrophy. The hypertrophy of the heart compensates for an increase in workload with an increase in cardiac output. However, excessive hypertrophy can result in cardiac dysfunction and substantially increases the risk of cardiac failure and mortality. The molecular mechanisms that regulate the development of cardiac hypertrophy and cardiac failure are not entirely understood. Traditionally, the G-protein Coupled Receptor (GPCR) and the downstream Mitogen-Activated Protein Kinase (MAPK) family of proteins have been implicated. However, elevated circulating and ventricular levels of several classes of cytokines also suggested that signaling by the downstream effectors of cytokine receptors, such as the Signal Transducers and Activators of Transcription (STATs), may be important. The aim of this thesis was, therefore, to characterize the involvement of MAPK and STAT pathways in regulating cardiac hypertrophy and cardiac failure. A function for MAPK and STAT signaling in regulating cardiac hypertrophy stimulated by the inflammatory cytokine IL-1Β was initially defined in primary cultures of neonatal rat cardiac myocytes. In this study, it was demonstrated that the chemical inhibition of ERK or p38MAPK was sufficient to inhibit IL-1Β-stimulated ANF expression. In contrast, simultaneous inhibition of both ERK and p38MAPK was required to ablate the hypertrophic morphology of cardiac myocytes treated with IL-1Β. These results demonstrated differential signaling from the MAPK isoforms in regulating the gene expression and morphological components of cardiac hypertrophy. In addition, it was revealed that IL-1Β treatment resulted in a delayed response (>60 min) in STAT3α tyrosine phosphorylation, which was subsequently shown to require the initial rapid activation of either ERK or p38MAPK. IL-1Β-stimulated STAT3 phosphorylation was also dependent on the de novo synthesis of secondary signaling molecules. The ablation of the STAT3 tyrosine phosphorylation by the inhibition of ERK or p38MAPK activity, correlated with the attenuation of IL-1Β-stimulated ANF expression, suggesting that signaling through STAT3α may be involved in regulating gene expression associated with IL-1Β cardiac hypertrophy

Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers. Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells. Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer. Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors. The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection. At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project. / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

The innate immune system is a critical first line of defense against invading pathogens. Innate immunity directly detects pathogens, sets up an appropriate adaptive response, and can directly kill pathogens. Drosophila may lack an adaptive immune response, but have a robust innate immune system with a variety of defense effector mechanisms. While the responses to bacteria, fungi, and RNA viruses have been well characterized, not much is known about the response to DNA viruses. My studies have set out to characterize the Drosophila immune response to a DNA virus, utilizing the large dsDNA virus, Invertebrate Iridescent Virus 6 (IIV-6). IIV-6 infection causes shortened lifespan, and in later stages of infection, flies present with abdominal swelling and iridescent blue color. Our objectives were to identify pathways flies use to protect themselves from IIV-6 infection, determine how this protection is mediated, and to identify any immune inhibitors that IIV-6 uses to suppress innate immune signaling. I have found that IIV-6 strongly up-regulates a class of stress proteins with unknown function, termed Turandots, after infection in vivo or in vitro. This induction is dependent upon viral replication, requires JAK-STAT activation, and activation of p38b MAPK. In addition, the unpaireds, which function as JAK-STAT ligands, are upregulated after IIV-6 infection in a p38b-dependent manner. Together, this data suggests that p38b activation leads to production of unpaired cytokines and activation of JAK-STAT signaling to induce Turandots. I have also found that IIV-6 infected cells secrete protective factors. This response is induced within 12 hours of IIV-6 infection, exosome-mediated, and provides robust protection to naive cells challenged with an mCherry-expressing strain of IIV-6. Additionally, IIV-6 inhibits two major immune responses in Drosophila, the IMD and Toll pathways. Stimulation of IIV-6 infected Drosophila S2* cells with either IMD or Toll stimulators results in very poor antimicrobial peptide responses. Yet, IMD and Relish are still cleaved upon stimulation in IIV-6 infected cells, indicating that the block is downstream. In support of this finding, IIV-6 infected flies respond very poorly to infection with the enterobacteria Erwinia carotovora carotovora compared to mock-injected flies.