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Molecular Prognostic and Predictive Markers in Colorectal Cancer
Navvabi, Nazila ; Pitule, Pavel (advisor) ; Rössner, Pavel (referee) ; Grubhoffer, Libor (referee)
in English Colorectal cancer is currently one of the three major causes of cancer-related death. In order to help clinicians to treat colorectal cancer, it is necessary to introduce more effective tools that will improve not only early diagnosis, but also prediction of the most likely progression of the disease and response to chemotherapy. This thesis aims to describe the most accepted biomarkers among those proposed for use in CRC divided based on the clinical specimen that is examined (tissue, feces or blood) along with their restrictions. In relation to my research, the thesis will also focus on alternative splicing that has emerged as an important regulator and potential treatment target in CRC. Alterations in gene expression leading to colorectal carcinogenesis results in dysregulated levels of nucleic acids and proteins, which can be utilized for the identification of modern, minimally invasive molecular biomarkers. One of the alternative splicing factors are MBNL proteins. The goal of my work that will be presented here was to analyze gene expression of the MBNL family of regulators of alternative splicing in various stages of colorectal cancer development, together with the MBNL-target splicing events in FOXP1 and EPB41L3 genes and tumor-related CD44 variants. The study was done using...
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Pathogenic Effect of Avian Leukosis Viruses in Chicken
Průková, Dana ; Geryk, Josef (advisor) ; Němečková, Šárka (referee) ; Grubhoffer, Libor (referee)
comparativestudyof pďhomorphologicalchangesin lymphoidtissuesinducedby ALVs of different subgroupspecificity, hasnot beenperformed.Usingthe chicken model we aimed at clarification of additional features,especially pathomorphologicaland immunological chara- cteristics of the wasting diseaseinducedby different strainsof ALV{. In orderto achievea propercompďison,alsoadditionalALV subgroup(A'B'D) wereincluded. on the otlrerhand, several retroviruseshave been found to induce kidney fumoÍs. MAv2 (N) is an avian nonacuteoncogenicÍetrovirusof subgroupB ALv. In chickens infec- ted in ovo or early afrerhatchingit induces,with high efÍrciency'multip|eclonal embryonic- type tumorsof kidney - nephnoblastomas(Wattsand Smith, 1980).The model of MAV2 (N)- induced chicken nephroblastomais basedon the assumedabiliý of MAV2(N) retrovirusto transformcells by insertionalmutagenesis,thatis, by a deregulationof expressionof geneshit by the proviral integration.This avian model ofnephroblastomarepresentsa valuabletool for identi$ing genesinvolved in themaligrant transformationof cells. It is assumedthatmacro- scopic nepfuoblastomasarise by clonal expansion of blastema cells in which MAV2 (N) provirus has deregulatedspecific genescontroling differenciation and proliferation @ajer et ď.' 2003). Summary ALVs have a large potential to...
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Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
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Posttranslational modifications in soluble recombinant therapeutical proteins secreted by lower aukaryotes: structure and function
Kumar, Vinay ; Bezouška, Karel (advisor) ; Šedo, Aleksi (referee) ; Grubhoffer, Libor (referee)
Virtat, Kunnr M.Sc Aint of the stud1, Aim of the studv The aim of this study was to understand the molecular mechanisnrs contributing to the production of soluble leukocyte receptors in the eukaryotic expression systenrs including the proper posttranslational modifications such as disulfide bond fornration and glycosylation. In order to achieve this goal, the following specific aims have been adopted for the study: l, t. To develop new methodologies for rapid and convenient assessnrent ofthe disulfide L I boltds in complex eukaryotic proteins using SDS polyacrylarride electrophoresis under nonreducing conditions, arrd nrass spectrometry. 2. To establish elements that are critical for the stability of soluble CD69 receptors expressed in the bacterial expression systenr 3. To develop methods for the production of glycosylated soluble CD69 proteins in lower eukaryotes (P i c h i a pa s to ri s) 4. To purify the proteins in sufficient quantities for biochemical and inrnrunological studies 5. To investigate, how disulfide bonding and glycosylation influeuces the stability of soluble CD69 receptors 6. To look at the carbohydrate binding activities ofthe produced proteins. 7. To evaluate the in vivo propefties of the produced proteins including their circulation half lifes in the blood of experiental animals, and...
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Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
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Nutritional biology of synanthropic mites (Acari: Acaridida)
Erban, Tomáš ; Smrž, Jaroslav (advisor) ; Grubhoffer, Libor (referee) ; Šustr, Vladimír (referee)
Ph.D. THESIS TITLE Nutritional Biology of Synanthropic Mites (Acari: Acaridida) ABSTRACT Several attempts to describe the nutritional biology of acaridid mites were undertaken, however full understanding of these processes remains incomplete. The objective of this Ph.D. thesis was to expand our knowledge concerning digestive physiology of stored product and house dust mites and to apply this knowledge to their nutritional biology. The research approach adopted in this Ph.D. thesis includes in vitro characterization of enzymatic activity in whole mite extracts (WME) and spent growth medium extracts (SGME), evaluation of the enzyme activities with respect to the gut physiological pH, enzyme inhibition experiments, in vivo localization of enzyme activities in the mite gut, determination of effects of nutrient or antifeedant additives in experimental diets on mite population growth and determination of the feeding preferences of synanthropic mites as assessed by in vitro and in vivo analyses. The gut contents of twelve species of synanthropic acaridid mites were determined to be within a pH range of 4 to 7 and showed a pH gradient from the anterior to the posterior midgut. The pH in digestive tract of synanthropic acaridid mites corresponds to the activity of proteases, α-glucosidases, α-amylases and...
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Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in the Czech Republic
Kybicová, Kateřina ; Hulínská, Dagmar (advisor) ; Žákovská, Alena (referee) ; Grubhoffer, Libor (referee)
The aim of this thesis was to assess the occurrence of two tick- borne bacteria, Borrelia burgdorferi sensu lato and Anaplasma pha- gocytophilum, in ticks, wild and domestic animals in the Czech Republic. In ticks, similar prevalence of both bacteria was ob- served. In rodents, the majority of infections were caused by B. afzelii while the infection with B. burgdorferi s. s. was also quite frequent. Infection with B. burgdorferi s. l. was more common in bank voles than in wood or yellow-necked mice. The prevalence of anti-Borrelia antibodies was higher in wood or yellow-necked mice than in bank voles. A. phagocytophilum was in a higher percentage of cases in the deer family and hares as compared to foxes and boars. We observed a similar prevalence of anaplasmosis in all domestic animals tested. We demonstrated that symptomatic dogs had a higher chance to be infected with A. phagocytophilum than asymptomatic dogs. Our findings suggest that the exposure to B. burgdorferi s. l. and A. phagocytophilum is common in vectors, reservoirs and hosts in the Czech Republic. Molecular and serological techniques for detection of these pathogens are also described in this thesis, including conven- tional PCR, nested PCR, real-time PCR with DNA quantification and melting curve analysis, RFLP analysis of the 5S-23S...
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