Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...

Clusters of Galaxies are amongst the largest gravitationally bound structures in our Universe and consist of thousands of galaxies. It is in these gigantic systems where Brightest Cluster Galaxies (BCGs) are found, the most massive galaxies in our Universe. A BCG, as its name indicates, is the brightest galaxy in a cluster. These enormous galaxies exhibit special properties, suggesting that they experience a different evolutionary path than a normal galaxy. It is widely accepted that their evolution involves merger events, when the BCG accrete another galaxy, as well as interaction events, like tidal stripping and/or removal of star-forming gas. However, the moment when those interactions happen in the life of the BCG and the extent of their impact on the BCG properties are still under discussion. This thesis aims to explore the later stages of BCG evolution by studying the merger/interaction fraction of BCGs in the local Universe. In particular, this research will explore the significance of correlations between the merger/interaction fraction with a variety of BCG properties (Metric Luminosity, α Parameter, BCG distance from the cluster center, BCG offset from the cluster mean redshift) and with a variety of cluster properties (cluster velocity dispersion, luminosity difference between the BCG and the second ranked galaxy in the cluster). The dependence of the merger/interaction fraction on the kinematics of BCGs is also investigated, using data for the BCG stellar velocity dispersion and for the local normalised velocity dispersion of galaxies within 50 kpc of the BCG. To accomplish these analyses, this thesis uses a sample of 432 BCGs at z ≤ 0.08 imaged as part of the Warpfire survey - an all-sky imaging and spectroscopic survey of BCGs in the nearby universe. Interacting and Non Interacting candidates are classified via a visual inspection of the residual images. This classification is performed by three independent people to ensure its robustness and to minimize classification bias. A merger/interaction fraction of fm/i = 0.220 ± 0.025 (stat) ± 0.040 (sys) at z ≤ 0.08 is found, with a lower limit of fmin ≥ 0.07 ± 0.01 (stat) ± 0.04 (sys). Significant correlations between the interaction status of BCG and its Metric Luminosity and α Parameter are also revealed. Specifically, the BCG merger/interaction fraction more than doubles in amplitude from ∼0.2 to ∼0.5 as the α Parameter increases from 0.4 to 0.9. However, those correlations do not appear to alter the Lm – α relationship, which remains robust against BCG interaction status. No significant correlation is found between the interaction status and the location of the BCG in the cluster, nor between the interaction status and the difference between the Metric Luminosity of the BCG and that of the second brightest galaxy in the cluster. However, it is found that BCGs with strong interaction residuals have slightly higher stellar velocity dispersions. Finally, the normalised velocity dispersion of galaxies within 50 kpc of the BCG is found to be lower than the normalised velocity dispersion around random galaxies in the outskirts of the cluster. The results of this thesis clearly indicate ongoing merger activity involving BCGs. The above results are consistent with idea that while BCG stellar accretion is not a dominant process at the current epoch it is not a negligible one either. These results also support a two phased growth model of BCG where the bulk of their stellar mass is assembled prior to z = 0.5 but still continues at a low level today.

Se realizó la presente investigación, con los objetivos de identificar y describir reacciones adversas a la vacuna con Bacilo de Calmette - Guerin en niños menores de 1 año e identificar y analizar los cuidados maternos en el hogar frente a las reacciones adversas. La investigación fue de tipo cualitativa, con abordaje estudio de caso. Los participantes fueron 15 madres que atienden a sus niños en el Centro de Salud Toribia Castro Chirinos y 7 enfermeras que laboran en dicho establecimiento; el muestreo se realizó por conveniencia y el tamaño se determinó con la técnica de saturación y redundancia, los datos se recolectaron con una guía de entrevista semiestructurada, validada por juicio de expertos y prueba piloto y se procesaron mediante el análisis de contenido. La ejecución de la investigación se tuvo en cuenta los criterios de rigor científico y los de rigor ético. Se obtuvieron como resultados 2 categorías: I: Reacciones adversas a la vacuna BCG. II: Cuidados maternos frente a las reacciones de la vacuna BCG. Llegando a la siguiente conclusión: la mayoría de madres con niños menores de 1 año que han sido vacunados con la vacuna BCG, manifiestan haber observado las reacciones tradicionalmente conocidas (nódulo, pústula, úlcera y cicatriz queloide), con ciertas particularidades cada una y con un tiempo de duración variado en muchos casos diferentes a literatura y a lo que el profesional de enfermería educa, siendo necesario que el profesional de enfermería los tenga en cuenta al momento de ejercer su rol educador en este tema.

Se realizó la presente investigación, con los objetivos de identificar y describir reacciones adversas a la vacuna con Bacilo de Calmette - Guerin en niños menores de 1 año e identificar y analizar los cuidados maternos en el hogar frente a las reacciones adversas. La investigación fue de tipo cualitativa, con abordaje estudio de caso. Los participantes fueron 15 madres que atienden a sus niños en el Centro de Salud Toribia Castro Chirinos y 7 enfermeras que laboran en dicho establecimiento; el muestreo se realizó por conveniencia y el tamaño se determinó con la técnica de saturación y redundancia, los datos se recolectaron con una guía de entrevista semiestructurada, validada por juicio de expertos y prueba piloto y se procesaron mediante el análisis de contenido. La ejecución de la investigación se tuvo en cuenta los criterios de rigor científico y los de rigor ético. Se obtuvieron como resultados 2 categorías: I: Reacciones adversas a la vacuna BCG. II: Cuidados maternos frente a las reacciones de la vacuna BCG. Llegando a la siguiente conclusión: la mayoría de madres con niños menores de 1 año que han sido vacunados con la vacuna BCG, manifiestan haber observado las reacciones tradicionalmente conocidas (nódulo, pústula, úlcera y cicatriz queloide), con ciertas particularidades cada una y con un tiempo de duración variado en muchos casos diferentes a literatura y a lo que el profesional de enfermería educa, siendo necesario que el profesional de enfermería los tenga en cuenta al momento de ejercer su rol educador en este tema.

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) is the leading cause of infectious disease-related death worldwide. The critical role of adaptive immunity in anti-TB host defence has been firmly established; thus, current efforts in developing novel vaccination strategies against TB are primarily focused on generating protective adaptive immunity at the infection site, the lungs. Innate immunity has not been a target for vaccination strategies against TB due to the belief that innate immune cells cannot exhibit memory-like characteristics which are known to be central to the long-lasting immunity created by vaccines. Also, the importance of innate immunity in anti-TB immunity has been overlooked. However, over 25% of individuals that are heavily exposed to M.tb clear infection without any detectable conventional T cell immune responses, suggesting a crucial role for innate immune cells in bacterial clearance. Interestingly, the early protection in these individuals is associated with their Bacillus Calmette-Guerin (BCG) vaccination status. Epidemiological studies have shown that BCG is capable of providing protection against numerous infections unrelated to TB in an innate-immune dependent manner. Such observations suggest that the innate immune system exhibits memory-like characteristics, capable of remembering the exposure to the vaccine and thereby responding in an augmented manner to future systemic infections. Nonetheless, it still remains unknown whether parenteral BCG immunization modulates the innate immune cells in the lung and airways, and if so, what role the trained innate immune cells play in early protection against pulmonary TB. Using a subcutaneous BCG immunization and pulmonary TB challenge murine model, we show that early protection against M.tb is independent of adaptive responses in the BCG immunized host. Our data suggest that enhanced early protection is mediated by the BCG-trained memory alveolar macrophages that we have shown to be functionally, phenotypically, metabolically, and transcriptionally altered following immunization. These novel findings suggest a significant anti-TB immune role for the innate immune memory established in the lung following parenteral BCG immunization and have important implications for the development of novel vaccination strategies against TB. / Thesis / Master of Science (MSc) / Pulmonary tuberculosis (TB) is a disease of the lung and is now one of the leading causes of human mortality worldwide. For more than eight decades, parenterally administered Bacillus Calmette–Guérin (BCG) vaccine has been globally used as the only approved vaccine against TB. Recently, it has also been observed that BCG vaccination provides protection against other diseases unrelated to TB and reduces childhood mortality in many developing countries where it is routinely administered to children shortly after birth. The mechanisms underlying the off-target protective effects of BCG vaccine remains largely under-investigated. In this project, we investigated how BCG vaccination enhances the immune system responses against TB and other unrelated infectious diseases. A better understanding of how the BCG vaccination modulates our immune system will provide us with the knowledge that will be useful in the development of more effective vaccination strategies against infectious diseases.

Le cancer superficiel de la vessie (CSV) présente un taux élevé de récidive (60%). De ces récidives 10 à 15% progresseront vers un cancer infiltrant beaucoup plus dangereux. La résection transurétrale (RTU) des tumeurs superficielles est souvent suivie d’immunothérapie intravésicale par le BCG (Bacille-Calmette-Guérin) afin de prévenir la récidive et la progression ; cependant ce traitement échoue dans 40% des cas. De plus, la sévérité des effets secondaires empêche plusieurs patients de tolérer un traitement complet. La prédiction de la réponse au BCG et le développement de traitements alternatifs s’avèrent donc nécessaires. Nous avons d’abord évalué la signification clinique de la présence de cellules dendritiques matures infiltrant la tumeur (CDIT) et de macrophages associés aux tumeurs (MAT) dans des CSV à bas risque, traités seulement par RTU. La présence de CDIT a permis d’identifier des patients à risque élevé de progression. Chez des patients à haut risque de récidive et de progression traités au BCG, nous avons observé que ceux qui ont un haut niveau d'infiltration par des CDIT ou des MAT ne répondaient pas efficacement au BCG. Dans un deuxième volet, j’ai exploré la possibilité d’utiliser d’autres agents théarapeutiques pouvant se combiner au BCG ou le remplacer pour stimuler la réponse antitumorale. Pour un tel rôle j’ai choisi les agonistes des Toll-like receptors (TLR). Les TLR, principalement exprimés par les cellules du système immunitaire et quelques cellules épithéliales, jouent un rôle important dans l’immunité innée en reconnaissant des motifs moléculaires conservés de pathogènes. J’ai d’abord montré que les TLR sont exprimés et fonctionnels dans des cellules urothéliales normales et tumorales. Par la suite, j’ai démontré que le poly(I :C), agoniste du TLR3, a un effet cytotoxique et antiprolifératif direct sur des lignées de cancer de vessie. Dans les cellules MGH-U3, il induit également la sécrétion de cytokines pro-inflammatoires et induit fortement l’expression des molécules du CMH de classe I alors que le BCG a très peu d’effet sur l’immunogénicité de ces cellules. Un essai d’inhibition de croissance tumorale utilisant le modèle de cancer de vessie murin MBT-2 a montré que l’utilisation combinée du BCG et du poly(I :C) inhibe très significativement la croissance tumorale alors que chacun des produits utilisés seuls n’avait pas d’effet significatif. Notre étude suggère que le poly(I :C) dû à ses effets anti-néoplasiques pourrait améliorer l’efficacité thérapeutique du BCG dans l’immunothérapie du CSV. / Non-muscle invasive bladder cancer (NMIBC) is characterized by a high rate of recurrence (60%). Ten to fiftheen % of the recurrences will progress toward muscle-invasive tumors, which are more dangerous. Transurethral resection (TUR) of non-muscle invasive tumors is frequently followed by intravesical immunotherapy using BCG (bacillus Calmette-Guérin) to prevent recurrence and progression but this treatment fails in 40% of cases. Moreover, the severity of the side effects prevents many patients to comply with the whole treatment. Tools to predict the response to BCG and the development of alternative treatments are therefore required. We first evaluated the clinical significance of the presence of tumor infiltrating mature dendritic cells (TIDCs) and of tumor-associated macrophages (TAMs) in low-risk NMIBCs treated only by TUR. The presence of TIDCs allowed the identification of patients that were at high risk of progression. In patients with NMIBCs at high risk of recurrence and progression treated with BCG, we observed that those with a high level of MAT or TIDC infiltration did not respond efficiently to BCG. In the second part of my work, I have explored the possibility to use other immunomodulatory agents to replace or complement BCG immunotherapy. I therefore selected toll-like receptors (TLRs) agonists for this purpose. TLRs, which are mainly expressed in immune cells but also epithelial cells, play an important role in the innate immunity by recognizing molecular patterns that are conserved between pathogens. I have first showed that TLRs are expressed and functional in normal and tumor urothelial cells. Then, I showed that poly(I:C), a TLR3 agonsist, has direct cytotoxic and antiproliferative effects on bladder cancer cell lines. In MGH-U3 cells, it induces the secretion of proinflammatory cytokines and expression of major histocompatibility class I molecules whereas BCG has little effect on the immunogenicity of these cells. A growth inhibition assay using the MBT-2 murine bladder cancer model showed that the combination of poly(I:C) and BCG inhibited very significantly the growth of bladder cancer cells whereas each product alone had no significant effect. Our study suggests that poly(I:C), due to its anti-tumoral effects, could improve the therapeutic efficacy of BCG for the immunotherapy of NMIBCs.

The aim of this thesis is to determine the strategy and partial steps that are needed to introduce the product on the market. The theoretical part defines the basic terms, which were described in accordance with professional literature related to this topic. The thesis describes SWOT analysis, BCG Matrix, market segmentation and Marketing Mix. In the practical part, the current state of the Marketing Mix is evaluated and it is pointed out how it should be newly set up correctly. The product development strategy was chosen based on Ansoff matrix. The market share of old brilliant watercolours gradually decreased, so the company took a step that innovated this product (saturation, pigment expansion and new packaging). Changes made to the product should have a positive impact on the volume of units sold and the gradual increase in market share. Furthermore, it is important that the company follows the new steps that are suggested based on the marketing mix. It is crucial that the company segment the market properly. In the Market Segmentation section, 11 categories are newly introduced. Brilliant watercolours are newly categorized as Creative, designed for a wide range of elementary art schools and creatives who are not satisfied with plain watercolours and products from ART category (professional products) are, in terms of price, inadequate to use. The new steps to introduce the product on the market include the costs that are reflected in the break-even point calculation. The resulting value is to show whether the payback time has been extended or shortened.

Die Familie der Mykobakterien setzt sich aus pathogenen und apathogenen Vertretern zusammen. In dieser Arbeit wurden 3 Mitglieder dieser Familie für Untersuchungen herangezogen: ihr prominentester pathogener Vertreter Mycobacterium tuberculosis, der Erreger der Tuberkulose, das als Impfstoff eingesetzte Mycobacterium bovis BCG, das durch Attenuierung aus dem Rindertuberkulose-Erreger Mycobacterium bovis hervorging und das apathogene Bodenbakterium Mycobacterium smegmatis. Ein Schlüssel zum Verständnis der Mykobakterien und speziell ihrer Widerstandsfähigkeit ist die Kenntnis ihrer komplexen Zellwand. Peptidoglycan als deren Bestandteil und insbesondere der mittels Undecaprenyl-Monophosphat bewerkstelligte Transport von Peptidoglycan-Vorläufern aus dem Cytoplasma an die Zelloberfläche steht dabei im Zentrum der Zellwandbildung. In M. tuberculosis, M. bovis BCG und M. smegmatis wurden Deletionsmutanten für die Undecaprenyl-Phosphokinase (Upk) hergestellt. Für M. smegmatis wurde gezeigt, daß die delta upk Deletionsmutante, in Übereinstimmung mit Deletionsmutanten homologer Gene in anderen Bakterien, eine erhöhte Sensitivität gegenüber dem die Zellwandsynthese hemmenden Antibiotikum Bacitracin aufwies. Überraschenderweise zeigte M. tuberculosis delta upk diesen Phänotyp nicht. Weiterhin ließ sich für M. smegmatis delta upk im Vergleich zum M. smegmatis Wildtyp Peptidoglycan an der Zelloberfläche in geringerem Maße nachweisen. Eindrucksvoll zeigte sich die Bedeutung der Undecaprenyl Phosphokinase in der gestörten Entwicklung von Biofilmen im Falle der M. smegmatis delta upk Mutante. Dies galt sowohl für in vitro Bedingungen als auch für ein, im Rahmen dieser Arbeit, neu entwickeltes in vivo Modell. Vergleiche von M. tuberculosis Wildtyp und M. tuberculosis Mutante auf der Ebene von Proteom- und Transkriptom-Analysen führten zur Identifikation eines zum mykobakteriellen Fettsäure-Synthese II (FASII) System gehörenden Operons, das im Falle der upk-Deletion verstärkt exprimiert wurde und damit möglicherweise einen Kompensationsmechanismus für die fehlende Phosphokinase darstellt. Eine reduzierte Persistenz von M. smegmatis delta upk in infizierten Makrophagen legte nahe, daß Upk bei mykobakteriellen Infektionen eine entscheidende Rolle für das Überleben der Bakterien und ihre Virulenz spielt. Dies konnte erstmals für M. tuberculosis im Rahmen von Maus-Infektionsversuchen gezeigt werden. M. tuberculosis delta upk ließ sich als neues Mitglied in eine Reihe von als growth in vivo (giv) klassifizierten Mutanten einreihen. Die Herstellung von Deletionsmutanten wird als Möglichkeit betrachtet, verbesserte Impfstoffe herzustellen. Die physiologische Konsequenz der Deletion sollte bestenfalls neben einer Attenuierung des Ausgangsbakteriums (gilt besonders für M. tuberculosis) eine Überexpression protektionsrelevanter Antigene zur Folge haben. Im Vergleich zum bestehenden Impfstoff M. bovis BCG führte die Impfung von Mäusen mit M. bovis BCG delta upk sowohl zu geringerer bakterieller im Anschluß an die Vakzinierung als auch zu einer verbesserten Langzeit-Protektion gegen Tuberkulose. / The family of mycobacteria is composed of pathogenic and apathogenic bacteria. This study was performed with 3 members of this family, the most prominent pathogenic member, Mycobacterium tuberculosis, the causative agent of tuberculosis, the vaccine strain Mycobacterium bovis BCG which was developed by attenuation of the bovine tuberculosis agent Mycobacterium bovis, and Mycobacterium smegmatis which is apathogenic and widely distributed in soil. A key to understanding mycobacteria and, especially, their resistance is to understand the complexity of their cell wall. Peptidoglycan is a major component of the cell wall and the transport of peptidoglycan precursors out of the cytoplasm to the bacterial surface by undecaprenyl monophosphate is central to cell wall synthesis. Therefore, deletion mutants of the undecaprenyl phosphokinase gene (upk) were generated in M. tuberculosis, M. bovis BCG, and M. smegmatis. In the case of M. smegmatis it was shown that a delta upk deletion mutant, as with deletion mutants of homologous genes in other bacteria, exhibited an increased sensitivity to the antibiotic bacitracin, indicating that cell wall synthesis was hampered. Surprisingly, M. tuberculosis delta upk did not exhibit this phenotype. Furthermore, a lower level of peptidoglycan was detected on the cell surface of an M. smegmatis delta upk mutant compared to M. smegmatis wildtype. Relevance of the undecaprenyl phosphokinase was demonstrated by impaired biofilm development in the case of the M. smegmatis delta upk mutant. This was observed in vitro as well as in vivo using an animal model which was newly developed in this thesis. A fatty acid synthase II (FASII) system related operon revealed by comparative proteome- and transcriptome-analyses comparing M. tuberculosis wildtype and M. tuberculosis delta upk mutant, and may reflect a compensatory mechanism for the loss of upk. Reduced persistence of M. smegmatis in infected macrophages suggested a decisive role of Upk in mycobacterial infection concerning survival and virulence of bacteria. This was later demonstrated to be true for M. tuberculosis in a mouse model. M. tuberculosis delta upk was, therefore, classified as a new member of the group of growth in vivo (giv) mutants. Construction of deletion mutants is a strategy to identify improved vaccines. Ideally, the physiologic consequences of a gene deletion would result in attenuation of the modified bacterium (especially in the case of M. tuberculosis) and overexpression of antigens relevant for protection. Compared to the existing vaccine M. bovis BCG, vaccination of mice with M. bovis BCG delta upk exhibited a lower bacterial load upon vaccination as well as an improved long-lasting protection against M. tuberculosis infection.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.

The main goal was to evaluate current market position of a selected Czech business unit within its industry. A selected joint-stock company is involved in an industry of fresh-water fishery. Main part of this thesis is focused on an analysis of fish market as a main foundation for analysing a market position of a future development in this industry.Other analysis processed for selected countries.