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Biocompatibility and immunocompatibility of polymers for gene therapy
Matyášová, Veronika ; Šírová, Milada (advisor) ; Tučková, Ludmila (referee)
Gene therapy is a potential strategy for treatment of diseases caused by a gene defect. Recent studies are involved particulary in the cure of diseases caused by single gene defect (cystic fibrosis, haemophilia, muscular dystrophy etc.). Our work is part of a project aiming at developing ex vivo non-viral gene delivery systems that could be used for the treatment of ocular and cardiovascular diseases. The gene vectors are biodegradable polymeric carriers based on poly-α-amino acids. These polyplexes should transfect target cells which are supposed to be seeded on polyimide membranes. The biodegradable polymer membrane will be implanted into the retina or used as a coating for cardiovascular prosthesis. As a cover of the implantable membranes we used polymerized methacrylamide-modified gelatin forming hydrogels and mediating a growth support for transfected cells. We focus on material bio- and immunocompatibility/immunoacceptability. The results indicated a very good bio- and immunocompatibility of the gelatin B hydrogel both in vitro and in vivo. The gelatin B hydrogel did not cause erythrocytes lysis, stimulation of proliferation (spontaneous or mitogen-induced) of mouse or human lymphoid cells, neither production of cytokines or NO in vitro. Histological examination following subcutaneous...
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Use of experimental animal models in chemotherapy induced peripheral neuropathy, possibilities for prevention and treatment
Hrzán, Lukáš ; Kubecová, Martina (advisor)
The topic of this diploma thesis is chemotherapy induced peripheral neuropathy. The first part is concerned with etiology, symptoms and diagnostics of peripheral neuropathy. The second part consists of a description of the most common antineoplastic drugs causing peripheral neuropathy. Different in vivo and in vitro models that are used in laboratory research to demonstrate the occurence of peripheral neuropathy are discussed. In the practical part, an experiment is described that was designed to evaluate whether the strain of CD1 mice can be a useful animal model to study chemotherapy induced peripheral neuropathy. Results of this experiment indicate that after 6 weeks of administration of cisplatin, paclitaxel and bortezomib, evidence of damage to the peripheral nervous system can be detected using neurobehavioral and neurophysiological tests. The final section includes information about current research in prevention and management of chemotherapy induced peripheral neuropathy. Latest recommendations for prevention and therapy of peripheral neuropathy in oncological patients are given.
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Study of transferrin as a marker of congenital disorders of glycosylation
Ondrušková, Nina ; Stiborová, Marie (advisor) ; Befekadu, Asfaw (referee)
Congenital disorders of glycosylation (CDG) represent a heterogeneous group of mul- tisystemic metabolic disorders which are caused by defects in biosynthetic pathways of glycoproteins. The screening test for N-glycosylation disorders is the analyses of sialylated isoforms of serum transferrin (Tf) by means of isoelectric focusing (IEF). Two distinct pathological IEF patterns of Tf are observed. A type I pattern is cha- racterized by a decrease of tetra- and an increase of di- and asialotransferrin, whereas a type II pattern shows in addition an increase of tri- and monosialotransferrin. The aims of diploma thesis were: 1) to evaluate reference range for spectrum of sialylated forms of Tf separated by IEF and 2) to perform biochemical and molecular analyses in three patients (P1-P3) with clinical suspicion for CDG. Serum and genomic DNA from three patients with clinical suspicion for CDG and family members of P1 were analysed. Sera from 99 healthy volunteers within the age range of 2-42 years served as a control group. Tf was analysed by IEF with direct immunofixation, SDS-PAGE and Western blot using specific antibody against human Tf (Dako). Profiles of Tf were quantified by AlphaEaseFC software (Alpha Innotech). Data were analysed by software STATISTICA 9.0 (StatSoft). TF a PMM2 genes were analysed...
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Impact of the Gastrointestinal System on Metabolic Control in Children with Type 1 Diabetes Mellitus - Gut Peptide Hormones
Huml, Michal ; Sýkora, Josef (advisor) ; Dědek, Petr (referee) ; Rušavý, Zdeněk (referee)
Our objective was to evaluate plasma levels of gut hormones and hormones associated with glucose metabolism in children with type 1 diabetes mellitus (T1DM), and to correlate plasma concentrations of gut hormones with blood biochemistry, markers of metabolic control and anthropometric parameters. A multiplex assay kit (LINCOplex® ) was used for the determina- tion of postprandial plasma levels of specific gut peptide hormones. Amylin, glucose- dependent insulinotropic polypeptide (GIP), active glucagon-like peptide 1 (GLP-1), active ghrelin, insulin, leptin, pancreatic polypeptide (PP), and polypeptide YY (PYY) were assessed prospectively in 55 subjects including 19 T1DM children (mean age: 13.4 years) and 21 healthy reference controls (mean age: 13. 4 years) and 15 patients with functional abdominal pain (FAP) (mean age: 10.5 years). In total, 440 plasma hormones samples were assessed in 55 patients. Entered data were examined using a non-parametric Wilcoxon's test. Further- more, statistically significant correlations were assessed by stepwise regression analysis. Our study demonstrated that the determination of specific postprandial gut hormones with the multiplex assay kit (LINCOplex® ) was highly efficient. Not only was a small amount of plasma sample (25µl) required for analysis, but also a...
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Local metabolism of glucocorticoids in female Prague hereditary hypertriglyceridemic rats
Klusoňová, Petra ; Pácha, Jiří (advisor) ; Kopecký, Jan (referee) ; Haluzík, Martin (referee)
11-hydroxysteroid dehydrogenase (11HSD1) is an oxidoreductase which catalyzes conversion of inactive 11-oxo steroid derivatives into active 11-hydroxy forms. 11HSD1 elevates intracellular level of active glucocorticoid (GC) hormones: cortisol in human tissues and corticosterone in rodents, therefore local level of active GCs can be set independently from systemic secretion driven by hypothalamo-pituitary-adrenal axis (HPA axis). Chronic systemic excess of GCs results in development of Cushing's syndrome which is characterised by central obesity and other metabolic disturbances. Despite normal serum levels of GCs, the patients with idiopathic obesity also develop metabolic syndrome. It was suggested that GCs could be elevated locally in target tissues due to enhanced 11HSD1 activity. This hypothesis was confirmed in transgenic rodent models. Prague hereditary hypertriglyceridemic (HHTg) rats represent a non-obese model of metabolic syndrome without genetic manipulations or specific mutations. The strain was bred by cross-mating of Wistar rat individuals with elevated serum levels of triglycerides (TGs). The strain exhibit hypertriglyceridemia and hypertension. When kept on high carbohydrate diet HHTg rats exhibit alterations in glucose homeostasis. Since there are no data that would describe...
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Phylogenetic analysis of Rieske dioxygenases large subunits genes in soil contaminated with jet fuel
Ptáček, Jakub ; Bořek Dohalská, Lucie (advisor) ; Poljaková, Jitka (referee)
The former military air-base Hradcany is among the most contaminated with organic pollutants localities in Czech Republic. Main cleanup strategy in the area is the bioremediation taking advantage on the natural potential of the autochthonous soil microorganisms to evolve catabolic pathways for in situ degradation of the pollutant. The diversity and abundance of the pathways, as well as the specificity and activity of the encoded enzymes are priority biotic factors determining the bioremediation efficiency. Main task of this work was to analyze the bacterial diversity in jet fuel contaminated soils based on key catabolic genes encoding the Rieske non-haem iron dioxygenases of the toluene/ biphenyl oxygenase branch. High molecular soil DNA was extracted and the sequences encoding catabolic genes were selectively enriched by hybridization to biotinylated oligonucleotides on magnetic microbeads with covalently bound streptavidin. Fragments of the genes for the -subunits of Rieske non-haem iron oxygenases were amplified and analyzed by restriction analysis, cloning and sequencing. Their evolutionary histories were inferred using the Neighbour-Joining and the maximum likelihood methods. The catabolic genes diversity in the actively bioremediated and highly polluted soil HRB was compared with the diversity in the...
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Comparison of sequence variations in genes of biotransfromation enzymes in some carcinoma
Turková, Lucie ; Tavandzis, Spiros (referee) ; Bóday, Arpád (advisor)
Xenobiotic biotransformation process and its capacity is crucial for xenobiotic chemicals elimination that may cause damage toward cell structures. The effectiveness of the enzymes included in this process depends on the gene variants that encodes them. The aim of this work was to compare certain polymorphisms of selected genes between cases and control groups. Studied polymorphisms were null genotypes of the glutathione S-transferase gene M1 and T1 and the insertion of TA dinucleotide in the promotor region of UDP-glucuronosyl transferase 1A1. The number of cases group was six included patients with colorectal, lung, prostate, breast, pancreatic and head and neck cancer. Total number of analysed individuals was 1 118 for cancer cases and 470 for healthy controls. The control group was divided into two groups, the first one was called general and the second one was called special included healthy individuals with no cancer history in their closest family members. Gilbert syndrome (GS) is caused by homozygous insertion of the TA dinucleotide in the TATA box of the gene UGT1A1 and it causes elevated bilirubin levels. Bilirubin is a potent antioxidant in human body, so the aim was to attest its protective effect toward cancer. We expected lower frequency of GS as a protective factor in the cases groups compared with controls. This hypothesis was confirmed in the breast cancer group (GS frequency 10,0 %) and pancreatic cancer group (GS frequency 11,1 %). In the general and special control groups the frequency of GS was 16,0 % and 15,4 % respectively. Although the other case groups show lower frequency of GS, the results weren´t statistically significant. Null GSTM1 genotype was observed with 50,4 % frequency in the general control groups and with 55,3 % frequency in the special control group. Neither the one of the cases groups hasn´t showed significantly lower percentage of null genotype. Despite expectation we observed statistically significant lower frequency of null genotype in the group of lung and pancreatic cancer group (37,4 % and 39,3 % respectively). According to this study, we can say that the lack of glutathione S-transferase M1 activity is not a risk factor for cancer development. Null genotype of GSTT1 wasn´t identified in both control groups at all. In case groups of breast and prostate cancer, there was only one individual carrying the null GSTT1 genotype. Statistically significant higher frequency of this polymorphism was observed in patients with colorectal cancer (9,7 %), lung cancer (17,2 %), pancreatic cancer (3,0 %) and head and neck cancer (15,9 %). In these groups the lack of glutathion S-transferase T1 activity might be considered as risk factor for cancer development. Nevertheless, for further verification it needs to take more investigation in this field, especially enlarge the number of patient in the case groups of head and neck, lung and pancreatic cancer.
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Analysis of financing small store in South Bohemia region
Pelc, Milan ; Říčka, Tomáš (advisor) ; Málek, Jiří (referee)
The first part of Master`s thesis focuses on defining the concept of small and medium sized company, a description of their meaning and the possibility of obtaining grants from EU structural funds and from government institutions. Within the options of grant will be prepared and submitted a grant application for a subsidized loan from Czech-Moravian Guarantee and Development Bank or from another entity. The second part of Thesis deals with the business plan. Thesis compares business plan from the franchisor with the reality that arose in building small store in Tabor and its operation in the first fifteen months. Based on the comparison may be designed to optimizing cost outlets of the store. The contribution of this Thesis is to look at a small retail business and it's financing. Such a complex view can help to similar retailers with orientation in the establishment and funding of a similar type of retail businesses. It will also be evaluated by franchise concept Náš grunt in the light of the real picture of financing its stores.
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