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Multiple forms of dipeptidyl peptidase IV and fibroblast activation protein in brain tumors
Matrasová, Ivana ; Šedo, Aleksi (advisor) ; Kupcová Skalníková, Helena (referee) ; Modrianský, Martin (referee)
Proteolytic enzymes are known to contribute to the initiation, development and progression of a number of diseases. Dipeptidyl peptidase IV (DPP-IV) and fibroblast activation protein (FAP) are serine proteases with the unique ability to cleave dipeptides containing - highly evolutionarily conserved - proline at the penultimate position of the N- terminus of substrates/biologically active peptides. FAP also exhibits gelatinolytic activity, which it exerts during extracellular matrix remodeling processes. Glial brain tumors (gliomas) arise from resident transformed glial cells, whereas brain metastases originate from circulating transformed extracranial tumor cells. Our previous work has described an increased expression of DPP-IV and FAP in high-grade glioma tissues. The presence of DPP- IV and FAP in brain metastatic tissues has not been described to date. The aim of this thesis was to describe the multiple forms of DPP-IV and FAP, and to describe their cellular origin and possible regulation in brain tumors. DPP-IV and its molecular MW and pI forms were expressed predominantly by transformed glial cells, whereas FAP and its MW and pI forms were expressed by transformed and stromal cells present in GBM and brain metastatic tissues. The spectrum of multiple forms of DPP-IV and FAP in GBM tissues and...
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Studies of intercellular interactions in tumours
Jechová, Alžběta ; Smetana, Karel (advisor) ; Skalníková, Helena (referee) ; Masařík, Michal (referee)
Beside tumor cells themselves, tumors consist of many non-malignantly transformed cellular elements and an extracellular matrix. This so-called tumor microenvironment, or stroma, significantly influences the biological properties of the tumor through intercellular interactions. In this thesis I have focused on the study of tumor-associated fibroblasts in squamous cell carcinomas of the head and neck, malignant melanoma and glioblastoma. The data show the presence of cells with mesenchymal characteristics, present even in the glioblastoma stroma, which could potentially have a positive effect on proliferative activity and invasiveness of glioblastoma cells. In malignant melanoma, the presence of keratinocytes should also be considered, as they are the major cells of the epidermis influencing tumor melanocytes. The conditioned medium from UVB irradiated keratinocytes and non-irradiated fibroblasts stimulates the invasion of malignant melanoma cells. Targeting the tumor stroma may be a new direction in oncological therapy, so we have focused on the influence of synthetic polyamine on the formation of myofibroblasts, which are an active part of the population of tumor-associated fibroblasts. The tested polyamine prevents the formation of myofibroblasts but has no effect on those already formed nor on...
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Proteomic analysis of cellular proliferation and differenziation: Model of neural stem cells and cancer cells
Skalníková, Helena ; Kovářová, Hana (advisor) ; Anděrová, Miroslava (referee) ; Bezouška, Karel (referee)
CoNcLUsIoNs In protein profiling of neural stem cells using 2-dimensional gel electrophoresis and mass spectrometry, constitutively expressed proteins in 66 protein spots were identified. Most of the individual protein species were related to RNA and protein metabolism, processing and turnover, including some chaperones and stress response proteins. Proteins involved in cellular organization (e.g. cýoskeletal proteins and annexins), metabolic proteins (mostly enrymes),cellular energetics,cell defenseand signallingfollowed in lower numbers. Proteins in 16 spots significantly regulated during neural differentiation were identified. Induction of levels of o-B crystallin, hnRNP Al and hnRNP AZIBI during differentiation and protein localization within neural cells were studied by westernblottingand immunocýochemistry. Using antibody microarrays, in neural stem cells an increase in GRK2 level and phosphorylationsof signalling molecules(CDKI|Z, PKC mu, PKCy, Erk5 and o-B crystallin) involved mostly in cellular proliferation were detected.On the contrary, in differentiatedneural cells levels of protein-phosphatase4, heme-oxygenase2, MEK3, RafB, pro-caspase 1 and phosphorylation of 40 kDa proline-rich Akt substratewere induced. In cancer cells after protein separationby ProteomelabrM PF 2D system, 8 proteins...
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Cytokine expression in regressive melanoma on porcine MeLiM model
Miltrová, Veronika ; Skalníková, Helena (advisor) ; Krulová, Magdaléna (referee)
Cutaneous melanoma is a very aggressive cancer with increasing incidence. It originates from transformed pigmented skin cells (melanocytes). The main risk factor for melanoma development is exposure to UV light and repeated sunburns. In approximately 10 % of cases, melanoma occurs on hereditary basis. Patients with cutaneous melanoma diagnosed in early stages have very good prognosis, with surgical resection of the primary tumour being mostly sufficient for treatment. In contrast, the advanced melanoma stages with metastases are often progressive and refractory to conventional therapies. Cutaneous melanoma is referred to as an immunogenic tumour that is frequently infiltrated by cells of the immune system. Tumours with immune cell infiltration show better prognosis. Spontaneous regression may occur. Over the last few years, progress has been made in the treatment of melanoma using checkpoints molecules (anti-CTLA-4 and anti-PD-1) to activate patients own immune system to recognize tumour lesions. In the tumour microenvironment, cytokines play an important role, enabling communication between cells and regulation of cell proliferation and migration and thus the tumour development. Cytokines (IL-2, IFNα) can be used in adjuvant therapy of melanoma. This work analysed levels of expressed cytokines in...
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Studies of intercellular interactions in tumours
Jechová, Alžběta ; Smetana, Karel (advisor) ; Skalníková, Helena (referee) ; Masařík, Michal (referee)
Beside tumor cells themselves, tumors consist of many non-malignantly transformed cellular elements and an extracellular matrix. This so-called tumor microenvironment, or stroma, significantly influences the biological properties of the tumor through intercellular interactions. In this thesis I have focused on the study of tumor-associated fibroblasts in squamous cell carcinomas of the head and neck, malignant melanoma and glioblastoma. The data show the presence of cells with mesenchymal characteristics, present even in the glioblastoma stroma, which could potentially have a positive effect on proliferative activity and invasiveness of glioblastoma cells. In malignant melanoma, the presence of keratinocytes should also be considered, as they are the major cells of the epidermis influencing tumor melanocytes. The conditioned medium from UVB irradiated keratinocytes and non-irradiated fibroblasts stimulates the invasion of malignant melanoma cells. Targeting the tumor stroma may be a new direction in oncological therapy, so we have focused on the influence of synthetic polyamine on the formation of myofibroblasts, which are an active part of the population of tumor-associated fibroblasts. The tested polyamine prevents the formation of myofibroblasts but has no effect on those already formed nor on...
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Proteomic analysis of cellular proliferation and differenziation: Model of neural stem cells and cancer cells
Skalníková, Helena ; Kovářová, Hana (advisor) ; Anděrová, Miroslava (referee) ; Bezouška, Karel (referee)
CoNcLUsIoNs In protein profiling of neural stem cells using 2-dimensional gel electrophoresis and mass spectrometry, constitutively expressed proteins in 66 protein spots were identified. Most of the individual protein species were related to RNA and protein metabolism, processing and turnover, including some chaperones and stress response proteins. Proteins involved in cellular organization (e.g. cýoskeletal proteins and annexins), metabolic proteins (mostly enrymes),cellular energetics,cell defenseand signallingfollowed in lower numbers. Proteins in 16 spots significantly regulated during neural differentiation were identified. Induction of levels of o-B crystallin, hnRNP Al and hnRNP AZIBI during differentiation and protein localization within neural cells were studied by westernblottingand immunocýochemistry. Using antibody microarrays, in neural stem cells an increase in GRK2 level and phosphorylationsof signalling molecules(CDKI|Z, PKC mu, PKCy, Erk5 and o-B crystallin) involved mostly in cellular proliferation were detected.On the contrary, in differentiatedneural cells levels of protein-phosphatase4, heme-oxygenase2, MEK3, RafB, pro-caspase 1 and phosphorylation of 40 kDa proline-rich Akt substratewere induced. In cancer cells after protein separationby ProteomelabrM PF 2D system, 8 proteins...
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Body fluid exosomes as potential carriers of Huntington’s disease biomarkers
Kupcová Skalníková, Helena ; Červenka, Jakub ; Bohuslavová, Božena ; Turnovcová, Karolína ; Vodička, Petr
Huntington’s disease (HD) is a hereditary neurodegenerative disorder characterized by a progressive motor, behavioural, and cognitive decline, ending in death. The cause of HD is an abnormal expansion of CAG repeats in HTT gene resulting in prolonged polyglutamine (polyQ) sequence in huntingtin protein (HTT). Huntingtin is a large protein (348 kDa) expressed ubiquitously through the body, with highest expression in the brain and testes. To study HD pathophysiology and to test experimental therapies, a transgenic HD minipig (TgHD) model expressing N-terminal part (N548-124Q) of human mutated huntingtin (mHTT) under the control of human huntingtin promoter was created in Libechov. Beside the mild neurological impairment, the TgHD boars show decreased fertility after 13th month of age.
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Quest for protein biomarkers of neural stem cells differentiation
Skalníková, Helena ; Halada, Petr ; Vodička, Petr ; Motlík, Jan ; Horing, O. ; Jensen, O. N. ; Gadher, S. J. ; Pelech, S. ; Kovářová, Hana
Understanding neurogenesis and neural cell differentiation presents a unique challenge for treatment of nervous system disorders. To gain more insight about molecular mechanism of differentiation of neural cells, we applied different proteomic approaches using classical 2-DE followed by MS and antibody microarrays. Based on 2-DE, profile of constituent proteins of neural stem cells and their differentiated progenies was estabilished at first and then the protein species that are significantly up or down regulated during the differentiation were selected. Differentiation of neural cells was accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage and redox regulation. Immunoblot verified changes of hnRNP A1, hnRNP A2/B1, RafB, heme-oxygenasy 2, GRK2 proteins and alphaB-crystallin (S45), CDK1/2 (Y15), PKC mu (S738+S742), proline-rich Akt substrate (T246) phosphorylations during differentiation.
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Protein fractionation and relative quantitation using PF 2D and ITRAQ for biomarker quest
Gadher, S. J. ; Skalníková, Helena ; Halada, Petr ; Řehulka, Pavel ; Chmelík, Josef ; Kovářová, Hana
ProteomeLab PF 2D System - Protein Fractionation in 2 Dimensions (Beckman Coulter, Fullerton, CA, USA) has been developed to fractionate complex protein mixtures by chromatofocusing in the first dimension followed by high-resolution non-porous silica reversed phase chromatography (RP LC) in the second dimension. Despite the high-resolution power of ProteomeLab PF 2D, UV-based quantitation could be compromised due to possible co-elution of several proteins into one fraction. Hence, we present an optimized protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and MALDI-TOF/TOF mass spectrometry to obtain quantitative data from peptides derived by tryptic digestions of intact proteins fractionated by ProteomeLab PF 2D technique. To demonstrate the feasibility of such an approach, protein expression patterns obtained from the ProteomeLab PF 2D fractionation of human T-lymphoblastic leukemia CEM cell line were utilised.
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Proteomika CDK inhibice v nádorových buňkách
Kovářová, Hana ; Skalníková, Helena ; Halada, Petr ; Strnad, M. ; Hajdúch, M.
In order to improve our understanding of the biochemical basis of the anti-cancer activity of olomoucine-derived synthetic cyclin-dependent kinase inhibitors (CDKIs) and to search for novel proteins associated with these biological effects we applied complex proteomic approaches. To analyse cellular responses to the CDKI we used two cancer models: the CEM T-lymphoblastic leukemia cell line representing hematological malignancy, and the A549 lung adenocarcinoma cell line as a solid tumor model. Cancer cells of these lines were cultured in both the presence and absence (controls) of the CDKI, bohemine (BOH). Cellular proteins of both of these lines were then extracted and fractionated using conventional two-dimensional gel electrophoresis (2-DE), and for the CEM T-lymphoblastic leukemia cell line we also used a 2-D liquid phase fractionation system ProteomeLab PF 2D ( Beckman Coulter). Computer-assisted data analysis of the resulting 2-D protein expression maps was applied to determine the similarity/dissimilarity of the maps and to select characteristic protein spots or bands based on the quantitative differences between BOH-treated and control cells. Many of these differentially expressed proteins were identified by mass spectrometry, since they represent candidate biomarkers of cancer cell responses to CDK inhibition and cellular pathways that are relevant to the anti-cancer activity of the CDKIs. Subsequently, we focused directly on these proteins in confirmatory studies using various techniques (including quantitative immunoblotting, immunocytochemistry and functional activity analyses) to demonstrate the validity of the proteomic results and extend our knowledge of the CDKI effects.
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