Charakteristika území Moravského krasu. Zpřístupněné jeskyně, propast Macocha. Historické, kulturní, církevní a technické památky. Materiláně technická základna Moravského krasu. Ochrana životního prostředí a speleoterapie. Průzkum povědomosti občanů ČR o CHKO Moravský kras. Swot analýza Moravského krasu

Colorectal cancer is the second most common cause of cancer death in Western Europe and North America. Current therapies are largely ineffective and are associated with considerable morbidity. Activating mutations in KRAS and BRAF genes are frequent in colorectal cancer, especially at later stages of the disease, and result in constitutive activity of the MAPK pathway, leading to increased proliferation and tumour survival. The MEK1/2 inhibitor AZD6244, that targets the MAPK pathway downstream of these mutations, has been tested as novel therapy for colorectal cancer. However, clinical trials have been disappointing due to an apparent intrinsic and/or acquired resistance to treatment. Mechanisms underlying this resistance have been studied using cell lines and tumour xenografts. However, the relevance of these data to advanced human colorectal cancer is unclear. One of the difficulties in testing and developing novel therapies for colorectal cancer is the lack of representative models of human disease. Thus, the initial aim of my PhD was to develop a method to culture human colorectal cancers ex vivo in order to use this as a platform for investigating response to AZD6244 and other therapies. These studies indicated that regardless of growth conditions, colonic tumour explants suffered extensive apoptosis in the first 24h in culture, which limited their application in drug response assays. Therefore, as an alternative to long term culture of human colorectal explants, I tested the effects of AZD6244 using acute treatments. Twenty three fresh colonic tumours were obtained from patients and treated for 1h with AZD6244 ex vivo in dose response studies. In all samples, MEK1/2 inhibition occurred within 1h of treatment. In one group of particularly sensitive tumours, the drug also had a distinct phenotypic effect. In these tumours, I found that the agent induced a dose-dependent decrease in proliferation and increase in apoptosis within 1h of treatment. Analysis of markers for this sensitivity indicated it was not clearly dependent of the presence of KRAS or BRAF mutations, which have previously been shown to confer sensitivity. Other markers of sensitivity / resistance were also examined. In addition to studies with AZD6244 alone, I examined the combined effects of this agent and aspirin in colon cancer cells lines and in tumour explants, with promising results. Whilst the use of fresh patient tumour tissue has some technical and logistical challenges, these data suggest that such methodologies are worthy of further investigation as a means to examine determinants of sensitivity and resistance to novel therapies, or their likely activity in combination.

This thesis is comprised of two parts. In the first part we study the influence of four frequently disputed genes on the susceptibility for developing prostate cancer, and in the second part we attempt to establish a basic understanding of the molecular genetic events in penile cancer. In a prostate cancer cohort we have investigated the relation of prostate cancer risk and single nucleotide polymorphisms (SNPs) in four different genes coding for the androgen receptor (AR), the vitamin D receptor (VDR), insulin (INS) and insulin receptor substrate 1 (IRS1). Despite strong biological indications of an involvement of these genes in prostate carcinogenesis, the results from different studies are contradictory and inconclusive. The action of the AR varies between individuals in part owing to a repetitive CAG sequence (polyglutamine) in the first exon of the AR gene. The results presented in this thesis show that in our cohort of prostate cancer patients the average number of repeats is 20.1, which is significantly (p<0.001) fewer repeats compared to healthy control individuals, where the average is 22.5 repeats. We find a 4.94 fold (p=0.00003) increased risk of developing prostate cancer associated with having short repeat lengths (≤19 repeats), compared with long repeats (≥23 repeats). In paper I we also study the TaqI polymorphism in the VDR gene, and find that it does not modify the risk of prostate cancer. In the INS gene we study the +1127 PstI polymorphism and find no overall effect on the risk of prostate cancer. However, we do find that the CC genotype is associated with low grade disease defined as having a Gleason score ≤6 (OR=1.46; p=0.018). In the IRS1 gene we study the G972R polymorphism and observe that the R allele is significantly associated with a 2.44 fold increased prostate cancer risk (p=0.010). The knowledge of molecular genetic events in penile cancer is very scarce and to date very few genes have been identified to be involved in penile carcinogenesis. We chose therefore to analyse the penile cancer samples using genome-wide high-density SNP arrays. We find major regions of frequent copy number gain in chromosome arms 3q, 5p and 8q, and slightly less frequent in 1p, 16q and 20q. The chromosomal regions of most frequent copy number losses are 3p, 4q, 11p and 13q. We suggest four candidate genes residing in these areas, the PIK3CA gene (3q26.32), the hTERT gene (5p15.33), the MYC gene (8q24.21) and the FHIT gene (3p14.2). The mutational status of the PIK3CA and PTEN genes in the PI3K/AKT pathway and the HRAS, KRAS, NRAS and BRAF genes in the RAS/MAPK pathway was assessed in the penile cancer samples. We find the PIK3CA, HRAS and KRAS genes to be mutated in 29%, 7% and 3% of the cases, respectively. All mutations are mutually exclusive. In total the PI3K/AKT and RAS/MAPK pathways were found to be activated through mutation or amplification in 64% of the cases, indicating the significance of these pathways in the aetiology of penile cancer.

Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.

<p>RAS is a small GTPase that helps to convert extracellular cues into intracellular actions. It is the most commonly mutated oncogene and is found in an active mutant form in 90% of pancreatic cancers. Therefore, study of how this protein is made and then how this protein signals in the cell could provide the foundation for novel approaches to treat RAS-driven malignancies.</p><p>First I demonstrate that the level of protein expressed from the gene KRAS, but not the highly homologous gene HRAS, is limited in mammalian cells by an abundance of underrepresented (rare) codons in the encoding mRNA. KRAS mRNA from both ectopic plasmids as well as from the endogenous cellular gene is subject to slowed translation due to these rare codons within its coding sequence. This has consequences for tumorigenesis, as replacement of the rare codons with commonly used codons accelerates RAS driven tumor growth. This may extend beyond HRAS and KRAS, as many other homologous gene pairs show a high divergence in codon usage and protein expression, suggesting that this could be a wider phenomenon used by mammalian cells to regulate protein levels.</p><p>Second, I demonstrate that RAS driven tumors partially depend on eNOS for growth. Using genetically engineered mouse models that recapitulate the spontaneous development of pancreatic cancer, I demonstrate that the protein eNOS is progressively upregulated as tumors develop. I then demonstrate that genetic ablation of eNOS partially blocks the development of preinvasive pancreatic lesions in these mice, and trends toward increasing survival in mice that develop lethal pancreatic adenocarcinoma. Furthermore, I then show that inhibition of eNOS using the clinically tested small molecule L-NAME can also slow the development of preinvasive neoplasia and nonsignificantly increase survival, although not to the level of eNOS genetic ablation. These findings are applicable to a clinical setting, as in conjunction with others I show that L-NAME treatment of human pancreatic cancer xenografts halves their growth, even when the main side effect of L-NAME, hypertension, is treated.</p><p>Together, these studies provide a better understanding of how RAS functions within the cell, and thus, ultimately, how RAS driven cancers may be treated.</p> / Dissertation

Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia. Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CXCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation. We conclude that MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.

This diploma thesis addresses recreation, relaxation and spending of free time in CHKO Moravsky kras, particularly in the Ostrov u Macochy community. In the area of the community there are many natural attractions, significant architectural objects and interesting tourist attractions. On the basis of field surveys of the territory and sociological surveys a 8,3 km long nature trail was designed on which 11 information boards are situated. The method of marking the trail, layout of the individual information panels, supporting infrastructure and possible sources of funding is addressed in the thesis. The nature trail was designed in compliance with the interest of the community, nature conservation bodies but also the users. All the materials and proposals can be used as the basis for implementation of the nature trail or structure serving for recreation in the community.

My thesis deals with some endangered species in the Training Forest Enterprise Křtiny and Moravian Karst, design certification of local forest nurseries logo regional product for sale, and also the importance for the local community. The work is focused on identifying endangered species of Training Forest Enterprise Křtiny, which are grown by the locals on their land in selected communities. Study deals with legislative restrictions in the management of endangered plant species. It explains the concept of the term "non-wood forest products", and its relevance to the forestry and biodiversity. In the work described conducted a field survey in which was collected seeds of selected endangered in the Moravian Karst for school forest enterprise Křtiny. Seeds were sown in a cold frame of Botanical garden and arboretum of Mendel University in Brno and made them germination tests. The last section describes the idea of certifing some forest nursery in the area of the Moravian Karst, giving the logo "MORAVSKÝ KRAS regional product." This tree nursery could selected endangered species grown and sold in order to increase the number of these plants in nature and the preservation of biodiversity.

In 2013, a faunistic research of the Carabidae tribe was carried out in the nature reserve of Mokřad pod Tipečkem (Wetland under Tipeček). The object of the nature reserve protection are waterlogged meadows and plant and animal societies typical for this environment. 12 ground traps were placed in the locality in four different areas (always on a mowed post, an unmowed one, the edge of a wood and the edge of a meadow with a wetland). Altogether, there were 34 species recorded with the total number of 328 individuals, among which there are two signifiant species, Leistus rufomarginatus with 3 individuals and Limodromus krynickii (2 individuals). Limodromus krynickii is recorded on the Red list of endangered species in the Czech Republic. In the above mantioned lokality, the most numerous species recorded were Pterostichus nigrita, Pterostichus niger, Abax parallelepipedus and Abax parallelus. Majority of the detected species are adaptace, prefering damp and umbral environment. The outcomes show that the area on the edge of a wood (area 3) is the most multifarious as to species and the number of individuals. On the other hand, areas 1 and 2 are not so multifarious.

This diploma thesis deals with description of the karst and its typical phnenomenons with emphasis on surface figures sinkholes. Further, it dicribes the airborne laser scanning (ALS) technology, data collection and processing procedure and utilization for the puropose of terrain depressions indentification based on the researches and processes of foreign experts. From these available studies, the most suitable methodics is chosen and applied to Digital Elevation Model of 5th generation (DEM 5G) data. The results of this aplication are compared with available sinkholes databases kept in the PLA Moravian Karst office and own terrain research.