Cystic Fibrosis (CF) is one of the most common heriditary diseases. This congenital condition, caused by CFTR gene mutation, affects gastrointestinal and respiratory system especially. The affection of respiratory system is considered the most serious life-threatening symptom. Pacients suffer from reccurent infections proceeding to the development of chronic inflammation and progressive pulmonary tissue destruction. A typical specific microoganism colonizating pulmonary tissue of thouse suffering from CF is recognised as Pseudomonas aeruginosa. Pulmonary infections caused by this microorganism are the most often cause of death in patients suffering from CF. Antiobiotics are the first-line therapy of this condition currently. Nevertheless, the need to find alternatives occurs due to antibiotics resistance development. Passive immunization by specific hen-egg-yolk antibodies against P. aeruginosa is a possible alternative. An observation of IgY influence on a bacterial adhesion to pulmonary epithelial cells required an appropriate model. In this context an appropriate adhesion testing method based on P. aeruginosa and pulmonary epithelial cells visualisation was searched. At first bacterial cells labelling was tested by CellTracker, resazurin, FITC and consequently PKH 26. P. aeruginosa fluorescent...

Germ-free animals have been used to study the effects of microbiota for several decades. In that time, numbers of differences from specific-pathogen-free (SPF) animals have been reported, including differences in absolute numbers or percentages of various immune populations, enormously enlarged coecum and lack of germinal centers. However, many of the crucial information about structural and functional differences in their secondary lymphoid organs still remains uncovered. With novel microscopical approaches, such as light sheet fluorescent microscopy, enabling 3D visualization of whole samples without processing them to a series of slides, and multicolor cytometry, allowing the characterization of numbers of cellular populations within a matter of seconds and in a highly quantitative manner, the uncovering of fundamental differences finally seems to be within reach. MHC II-EGFP knock-in mouse model brings the advantages of a fluorescent protein expressed in physiological histological contexts into both fields. Lymphoid and other tissues can be visualized microscopically without the need of staining (even in vivo). Information about the expression of both plasma membrane-localized and intracellular MHC II in various tissues could be acquired directly. Combining MHC II-EGFP knock-in mouse model with...

The plot of this PhD thesis is dedicated to investigation of the molecular pathways and events and their disruptions in the gastrointestinal tract (further abbreviated as GIT). The major role in this part plays the Wnt signaling pathway. This marvelous multipurpose machinery is responsible for epithelia renewal from stem cells (SCs) in the stomach and intestine, and for correct zonation and function of hepatic units. Of note, the Wnt pathway directs also development of embryo as well as homeostasis of many tissues apart from GIT in all metazoans, thus its flawless function is indispensable from one's origin to death. The main part of the thesis follows canonical Wnt signaling in its physiological condition and, in contrast, with pathological disturbances. This issue can be taken by variety of means as it is described in attached publications. The first publication deals with searching for new participants of Wnt signaling and their functions and describing unique markers of SCs in the intestine. Troy, the member of tumor necrosis factor receptor (TNFR) superfamily, was identified as a novel marker of intestinal SCs by probing microarray data from chromatin immunoprecipitation obtained in cultured colorectal cancer cell lines. Moreover, we found that Troy is a Wnt target gene inhibiting the...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

The development of immune response accountable for the ability to control Cryptosporidium muris TS03 infection was studied using immunocompetent and various types of immunodeficient mouse models. Subsequently the immune response was characterized by analysis of leukocyte infiltration and cytokine production in gastric epithelium. Moreover, the potentiality of immunocompetent mice to develop effective immune response to C. andersoni LI03 infection with consequent protection to consequent infection of the same mice with C. muris TS03 was also studied by monitoring oocysts shedding, leukocyte infiltration of the gastric mucosa and cytokine production in ex vivo cultures of splenocytes.

Colorectal cancer is one of the most common cancer types worldwide. Both genetic and epigenetic alterations play a critical role in its initiation and progression. One of the genes frequently epigenetically silenced or lost in many types of human cancer is tumor suppressor gene Hypermethylated in Cancer 1 (HIC1). It encodes for transcriptional repressor regulating its target genes directly or indirectly. Twelve genes whose expression is repressed by HIC1 have been identified to date. These genes encode for transcription factors, cell cycle and apoptosis regulators or proteins involved in angiogenesis as well as cell migration and invasiveness. Employing mouse embryonic fibroblasts upon Hic1-conditional knockout we have revealed six novel genes potentially repressed by Hic1 including Toll-like receptor 2 (Tlr2). Here we show that Tlr2 is one of the Hic1 target genes and that Hic1 inactivation in the intestine leads to increased Tlr2 production. Moreover, enhanced inflammatory response upon chemical-induced colitis as well as increased tumor formation in ApcMin mice was observed in Hic1-deficient mice. Expression profiling in human fibroblast upon HIC1 knockdown revealed increased expression of another potential target gene, transcription factor E2F7. Our study describes a new relationship between HIC1 and...

Colon cancer causes approximately seven percent of all cancer-related deaths in the world and presumably due to modern lifestyle, it is also one of the most frequently diagnosed cancers. The inefficiency of standard treatment indicates the need for intensive research of molecular mechanisms of cancer development. The canonical Wnt signaling pathway is essential for maintenance of the progenitor phenotype of stem cells in crypts of the intestine and controls repopulation of the epithelia, in physiological conditions. However, aberrant activation leads to tumor formation. Although Wnt signaling in cancer has been subjected to thorough investigation, there is still a lot of questions concerning further branching of the pathway. As a model of Wnt/β-catenin triggered colorectal cancer, we use mice with mutated APC, which is the tumor suppressor involved in this pathway. Previous expression profiling of the intestinal tumors from relevant mice revealed two transcription factors: ETV4 and MSX1 which are significantly overexpressed in cancer cells. In this project we elucidate whether the overexpression is really tumor restricted and Wnt dependent or there is a crosstalk with another signal transduction pathway. We investigate the function and regulation of these transcription factors by synthetic reporter assays,...

AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...

The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....