The activation of bone marrow-derived mast cells (BMMCs) induces a number of cell processes such as degranulation, proliferation and cytoskeleton rearrangements. Although microtubules are important in these processes, molecular mechanisms that control changes in microtubule organisation during cell activation are unknown. Activation of BMMCs can be achieved in several ways. Under physiological conditions, the aggregation of IgE receptors (FcRI) on the surface of BMMCs leads to the initiation of specific signaling pathways. Cells can be also activated nonspecifically by a tyrosine phosphatase inhibitor pervanadate, or by thapsigargin that inhibits Ca2+ ATPase pumps located on the endoplasmic reticulum. In this diploma thesis it was found out that rapid morphological changes can be monitored when BMMC are immobilised on the fibronectin before their activation. It was proved that specific and nonspecific activation events lead to microtubule reorganization, as well as to generation of a large number of microtubule-dependent protrusions. In the course of FcRI aggregation, generation of microtubule protrusions depends on the activity of Src family protein tyrosine kinases and on the intracellular Ca2+ concentration. STIM1, an endoplasmic reticulum Ca2+ sensor, which participates in the activation of...

Microtubules (MTs) are highly dynamic structures essential for the spatio-temporal intracellular organization and transport, signal propagation, cell differentiation, motility and division. To perform these roles, MTs create arrangements capable of fast and precise adaptation to various signals. MTs are under the control of many factors regulating MT nucleation, stability and dynamics. Bone marrow-derived mast cells (BMMCs) are important immune system cells, which can cause serious diseases if their functions are deregulated. Although MT reorganization during BMMC activation is well established, the molecular mechanisms that control their remodelling are largely unknown. In the presented thesis we functionally characterised GIT1/βPIX signalling proteins, PAK1 kinase, and Ca2+ signalling in the regulation of MT nucleation in BMMCs and other cell types. We also elucidated the function of miltefosine (hexadecylphosphocholine), a promising candidate for the treatment of mast cell-driven diseases. We found that GIT1/βPIX signalling proteins are γ-tubulin-interacting proteins associating with centrosomes in BMMCs. MT nucleation is positively regulated by GIT1 and Ca2+ , whereas βPIX is a negative regulator of MT nucleation in BMMCs. Cytosolic Ca2+ affects γ-tubulin properties and stimulates the...

As tapping energy from wind expands rapidly worldwide, it is a common procedure to locate a practicable site to extract energy from abundant wind flow by building wind farms. Comprehensive understanding of wind resource at a site   is important to perform the main activities say, wind flow modeling, wind   turbines micro siting, annual energy yield calculation and cost of energy   estimation. Wind measuring campaigns involve using of measuring instruments   such as meteorological tower instrumented with anemometers, wind vanes and   temperature sensors; remote sensing devices such as SoDAR, LiDAR. These   meteorological devices provide detailed information on wind behavior with   respect to the height, time and temperature. These systems were proven in   providing promising wind measurements even though they are susceptible to   certain weather conditions. The   study progressed by focusing on the wind behavior at three locations to   investigate the possible factors that varies the wind character. A location   with one met mast and two AQ500 SoDAR systems was considered for this project   where one AQ500 is 800m away and other AQ500 is 5515m away from the point of   Met mast. The location is contemplated as a decent approach to the spatial   analysis of the wind resource as there is a large scope to analyze the wind   character between two nearby sites and two faraway sites. Monostatic 3-beam   SoDAR systems from AQ System, Sweden and 100m meteorological tower with   instruments are used in this project work for collecting the wind data.   One-year worth of wind data at standard 10min intervals has been collected   from the three systems. This report outlines the theoretical description of project location, AQ500 SoDAR and Met mast. A detailed explanation of the data quality control and filtering methods are discussed along with respective reasons. The conclusion is drawn after performing the statistical analysis between wind speed and other parameters such as turbulent intensity, wind direction, thermal stability and temperature. Mat lab is used for computing and analyzing the wind data from three systems.

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

Human mast-cell chymase (EC 3.4.21.39) is a chymotrypsin-like serine protease that is stored in and released from mast-cell granules. This enzyme has been expressed in Pichia pastoris by homologous recombination of the cDNA coding for the mature active chymase into the Pichia genome. Cells producing the highest levels of recombinant human chymase were selected by activity screening and they were grown in a fermentor. Methanol induction resulted in the secretion of active chymase into the Pichia growth media and increasing levels of enzyme were detected in the media for 5 days. Active enzyme was purified from the culture media with a 22 % yield of activity by a simple two-step procedure involving hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media.

La peau est un « avant poste » fascinant du système immunitaire. Elle forme une barrière entre l'environnement extérieur et l’organisme. Elle est aussi le point d'entrée pour les agents pathogènes, contre lesquels le système immunitaire organise des réponses adaptatives. Les acteurs de l'immunité innée de la peau contrôlent l'invasion des pathogènes et perçoivent également des changements environnementaux physiques et chimiques directs. Plusieurs composants du système immunitaire, tels que des cellules dendritiques (DCs), les macrophages (MΦ) et les mastocytes (MCs), participent à l'éradication des pathogènes et à l'initiation des réponses mémoires adaptatives. Ce qui permet une mobilisation rapide des cellules T effectrices ainsi que la sécrétion des anticorps par les cellules B à la suite d’une seconde exposition aux agents pathogènes. Les MCs qui sont des cellules résidentes du derme, jouent un rôle déterminant dans la libération de signaux d’alertes et sont classiquement considérés comme des cellules effectrices de la réaction allergique cutanée liée à l'IgE. Plusieurs observations récentes indiquent que les MCs seraient aussi impliqués dans les processus immunorégulateurs lors de l'initiation des réponses immunitaires adaptatives, dans le maintien de la tolérance périphérique aux composants de la peau et dans la régénération de la peau au cours des processus de cicatrisation. Cependant, les interactions entre les MCs et d'autres cellules immunitaires innées et adaptatives recrutées dans des conditions inflammatoires cutanées n'ont pas été élucidées en détail. Dans ce travail, nous décrivons l'utilisation d'une nouvelle souris possédant des MCs fluorescents (RMB), dans laquelle nous avons marqué les MCs FcεRI+ avec un marqueur fluorescent rouge tomato (TdT) et avec un système d'ablation conditionnelle basé sur l'expression concurrente du récepteur de la toxine diphtérique (DTR). Avec ces souris RMB, nous avons visualisé la dynamique des MCs et nous avons suivi les interactions entre les MCs et les lymphocytes T régulateurs (Tregs) après l'activation des MCs par l'IgE, dans une réaction inflammatoire typique de l'anaphylaxie cutanée passive (PCA). Dans un second volet d’étude, nous avons évalué le rôle des MCs lors d'un modèle expérimental de la greffe de peau de l'oreille, afin de révéler leur influence dans la cinétique de rejet ou prise de greffe du transplant. Nous avons constaté que 1) l'activation et la dégranulation des MCs induites par le pontage du récepteur FcεRI via des IgE couplées à un antigène multivalent sont les seules responsables de la réaction de PCA, et induisent le recrutement de Tregs ayant une grande motilité sur le site de l'inflammation. Nous avons constaté dans ces conditions, que les MCs restent immobiles, et que les Tregs établissent des contacts dynamiques avec les MCs dans le derme. 2) En outre, nous avons mis en place un modèle pour identifier les paramètres moléculaires de l'interaction MC-Treg et avons constaté que le complexe de l'antigène avec l'IgE peut être présenté aux Tregs en association avec les molécules du complexe majeur d'histocompatibilité de classe II, permettant la formation des contacts stables MC-Treg. 3) En utilisant un modèle de transplantation de la peau in vivo, nous avons montré que l'ablation conditionnelle des MCs conduit à une accélération du rejet du greffon dans le cas d'une transplantation en présence d’une disparité d’antigènes d’histocompatibilité mineurs depuis une souris mâle sur une souris femelle. Nous avons également constaté un impact inattendu de l'ablation des MCs dans la greffe de peau en l’absence de disparité antigénique d'une souris femelle sur une souris femelle, conduisant à un rejet rapide. Les MCs semblent donc être essentiels pour la cicatrisation et la régénération tissulaire après greffe. (...) / The skin is a fascinating outpost of the immune system. It performs a barrier function between the outside environment and the inner body and is also a port of entry for pathogens against which the immune system mounts adapted responses. The skin innate immune defenses control pathogen invasion and perceive also direct physical and chemical environmental changes. Several component of the immune system such as dendritic cells (DC), macrophages (MΦ) and mast cells (MC) participate in initial pathogen clearance and in initiating adaptive memory responses, allowing rapid mobilization of effector T cells and secretion of B cellderived antibodies after secondary pathogen challenge. MCs residing in the dermis exert a determinant alert function through the liberation of various factors and are classically considered as effector cells in the IgE-mediated cutaneous allergic reaction. As emerging now, MC are also involved in immunoregulatory processes during the initiation of adaptive immune responses, the maintenance of peripheral tolerance to skin components and skin regeneration during wound healing. Yet, the crosstalks between MCs and other innate and adaptive immune cells recruited during cutaneous inflammatory conditions have not been elucidated in detail. Here, we report the use of a novel Mast cell fluorescent reporter mouse (RMB), in which we tagged FcεRI+ MCs, with red fluorescence marker tomato (Tdt) and with a conditional ablation system based on concurrent diphtheria toxin receptor (DTR) expression. Using these RMB mice, we visualized MC dynamics and monitored MC interactions with regulatory T lymphocytes (Tregs) after IgE-mediated activation of MCs, in a typical passive cutaneous anaphylaxis (PCA) inflammatory reaction. Using another setting, we further assessed the role of MC during experimental ear skin grafting to reveal their potential influence in skin grafting and rejection. We found that 1) the activation and degranulation of MCs induced by FcεRI crosslinking by multivalent IgE is solely responsible for the PCA reaction and induces the recruitment of highly motile regulatory T cells (Tregs) to the site of inflammation. In these conditions, we found that MC remain sessile and Tregs establish dynamic contacts with MC in the dermis. 2) Further we set up a model system to reveal the molecular requirement for MC-Treg interaction and found that antigen complexed with IgE were able to be presented to Treg in association with major histocompatibility complex class II molecules allowing the formation of stable MC-Treg contacts. 3) Using in vivo skin transplantation model, we showed that conditional ablation of MCs leads to an acceleration of skin transplant rejection in sex-mismatched model (male skin transplant to female). We also found an unexpected impact of MC conditional ablation in sex-matched skin graft (female skin transplant to female) leading to rapid rejection, implying that MCs are essential for the wound healing reaction and the regeneration of tissue continuity after grafting. The aforementioned results point out to an important immunoregulatory role of MC beyond their classically described activator functions in inflamed tissues. The fact that MC constantly interact with Treg during inflammatory processes suggest that MCs could participate in skin homeostasis by exerting tolerogenic functions. These functions remain to be elucidated at the molecular level as presented in the discussion.

Les mastocytes humains (MC) sont des cellules tissulaires d’origine hématopoïétique impliquées dans une série de processus physiologiques et pathologiques. Les recherches sur les MC ont été entravées pendant longtemps en raison de l'accès limité à des populations pures de ces cellules. Nous avons établi une nouvelle lignée humaine de MC, ROSAKIT WT, dont les propriétés sont similaires à celles des MC primaires, constituant un nouvel outil pour la recherche sur les fonctions des MC humains, et permettant le criblage à haut débit de thérapies anti-allergiques. Les MC sont impliqués dans les mastocytoses, où ils s’accumulent pathologiquement dans divers tissus. Bien que la plupart des cas de mastocytoses systémiques (SM) sont chroniques et indolents, les patients atteints de SM avancée (SM agressive; ASM, et leucémie à mastocytes; MCL) ont un mauvais pronostic, car la plupart des thérapies disponibles ne sont pas curatives. Afin de mieux comprendre la physiopathologie des formes avancées de SM et pour trouver de nouvelles approches pour le traitement, nous avons profité de la disponibilité des cellules ROSAKIT WT pour établir un nouveau sous-clone, la lignée cellulaire ROSAKIT D816V, représentant un équivalent des cellules néoplasiques s’accumulant dans les SM. L'utilisation de cette lignée et de cellules des patients nous a permis d’identifier BRD4 comme une nouvelle cible thérapeutique dans les ASM et les MCL. Nous avons démontré que les MC néoplasiques de patients avec ASM expriment des quantités substantielles de BRD4. Fait intéressant, nous avons aussi démontré que les lignées cellulaires HMC-1 et ROSAKIT D816V expriment aussi BRD4, et que leur prolifération est inhibée par un shRNA BRD4-spécifique. En outre, nous avons montré que le médicament JQ1, inhibiteur de BRD4, induit une inhibition de la croissance et une apoptose dose-dépendante dans les mêmes cellules. De plus, nous avons démontré que JQ1 supprime également la prolifération des MC néoplasiques primaires de patients atteints d’ASM ou de MCL à de faibles concentrations. Enfin, nous avons observé que la midostaurine (PKC412) et l’acide rétinoïque tout-trans (ATRA) coopèrent avec JQ1 pour induire des effets inhibiteurs synergiques sur l’inhibition de la survie des mêmes cellules. En conclusion, nos résultats représentent une avancée sur ce qui était précédemment connu sur l’implication de BRD4 dans les mastocytoses et nous ont permis d'identifier cette protéine comme cible thérapeutique prometteuse dans le traitement des formes avancées de SM. / Human mast cells (MCs) are hematopoietic stem cell (HSC)-derived, tissue-resident, multifaceted cells involved in a myriad of physiological and pathological processes. Researches on MCs have been hampered for a long time, due to limited access to pure populations of these cells. We have established a new human MC line, ROSAKIT WT, whose properties are similar to those of primary HSC-derived MCs, providing a novel tool for research on human MC functions, and enabling the high-throughput screening of anti-allergic therapies. Among others, MCs are involved in a group of diseases termed mastocytosis, where they accumulate pathologically in various tissues. Although most cases of systemic mastocytosis (SM) are chronic with an indolent course, patients with advanced SM (aggressive SM; ASM, and mast cell leukemia; MCL) have a reduced life expectancy and a poor prognosis, since most of the therapies already available are not curative. In order to better understand the pathophysiology of advanced SM and to. find new approaches for treatment, we took advantage of the availability of the ROSAKIT WT cells to establish a new subclone, the ROSAKIT D816V cell line, representing a paradigm of the neoplastic cells accumulating in SMUsing these malignant cell line and patients’ cells, we identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as a novel drug target in ASM and MCL. Indeed, we demonstrated that neoplastic MCs from ASM patients expressed substantial amounts of BRD4. Interestingly, we then demonstrated that HMC-1 and ROSAKIT D816V cell lines express BRD4, and that their proliferation is inhibited by a BRD4-specific shRNA. Moreover, we showed that the BRD4-targeting drug JQ1 induced a dose-dependent growth inhibition and apoptosis in the same cells. In addition, we demonstrated that JQ1 suppressed also the proliferation of primary neoplastic MCs of patients with ASM or MCL at low concentrations. Finally, we reported that midostaurin (PKC412) and all-trans retinoic acid (ATRA) cooperated with JQ1 in producing synergistic inhibitory effects on the survival of HMC-1 and ROSA cells. Together, our data represent a significant advance over what was previously known on the involvement of BRD4 in mastocytosis and identify this epigenetic reader bromodomain-containing protein as a promising drug target in advanced SM

1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...

CHAPTER 1 Aggregation of receptors for IgE (Fc RI) causes mast cells and basophils to release preformed contents of granules, including histamine and a variety of enzymes. This process, called degranulation plays a central role in allergic reactions. Methods to study this process are to create multivalent ligands which can interact with the receptors and, in turn, lead to aggregation of the receptors. We prepared a series of fluorophore-labeled divalent and trivalent antigens to study the degranulation of mast cells. Trivalent antigens proved to be much better stimulators for degranulation of mast cells than divalent antigens. These results indicate that aggregates formed by trivalent antigens are more complicated than those of divalent antigens. CHAPTER 2 Membrane-active antibiotics include antimicrobial peptides (AMPs) and a class of amphiphilic steroids termed ceragenins. Recent studies of membrane-active antibiotics show that cationic, facially amphiphilic molecules could disrupt bacterial membranes. It was found recently that some antibiotics, including AMPs and ceragenins, may share both antibacterial and antiviral activity. We prepared a series of ceragenins to optimize the antiviral activity of ceragenins against vaccinia virus (VV). The results show that ceragenins exhibit potent activity against VV, protect keratinocytes against VV-mediated cell death, and preferentially target the virus. It also shows that antibacterial and antiviral activities do not correlate with each other. Although ceragenins show good antiviral activity against VV, the mechanism for this activity still remains unclear.

Wind energy is rapidly expanding worldwide, and it is common practice to maximize production by selecting sites with higher wind potential. To perform critical operations such as wind flow modeling, wind turbine micro placement, annual energy yield calculation, and cost of energy estimation, a thorough understanding of a site's wind resource is required. The present study examines data from three independent wind measurement systems to see how measured data depends on the choice of the measurement system and how this might forecast the wind resource and, consequently, the energy output of a potential wind farm.  The present analysis uses three measurement units, one meteorological mast (met mast), and ground-based AQ510 Sound Detection And Ranging (SoDAR) & SoDAR and ZX 300 Light Detection And Ranging (LiDAR) devices to capture wind data for nearly a year. This study describes the operating concept of remote sensing devices such as AQ510 SoDAR and ZX 300 LiDAR, the linear regression relationship between wind speed measured on the Met Mast versus SoDAR, Met Mast versus LiDAR, and SoDAR versus LiDAR. Additionally, an understanding of stratification for this potential wind farm’s site is explored for specific days during spring, summer, and winter.  The results of the intercomparison study among Met Mast, SoDAR & LiDAR show quite a good relationship between the different measurement systems, being the correlation coefficient between the mast and the LiDAR measurements being slightly larger than between the mast and the SoDAR measurements. Comparison during the stability and instability regimes show a larger difference in some cases. Python and MS Excel are used to build data filtering procedures, the Richardson number, and comparison computations.