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Studies of the Mouse polyomavirus: properties of the minor structural proteins, requirements for virion productive trafficking to the nucleus and observed side effects of DNA transfection.
Huérfano- Meneses, Sandra ; Forstová, Jitka (advisor) ; Mělková, Zora (referee) ; Reiniš, Milan (referee)
In this thesis, we aimed to investigate the role of Mouse polyomavirus (MPyV) minor structural proteins, VP2 and VP3 in the viral life cycle as well as to study specific aspects of the productive trafficking of the virus. The thesis is divided into 3 chapters as follows: chapter 1 addresses questions about the interaction of the minor structural proteins, VP2 and VP3, of MPyV with host cells and their role during late stages of virus infection. In the chapter 2, aspects concerning the role of late endosomes, recycling endosomes and actin cytoskeleton in MPyV productive infection are addressed and in the chapter 3, results of studies of interferon (IFN) responses induced after DNA nucleofection are presented. The study in chapter 3 was preceded by a microarray analysis primary performed to reveal cell responses to the presence of the minor proteins within host cells. We showed that both MPyV minor structural proteins, VP2 and VP3, when produced individually (without VP1) are highly cytotoxic, inducing fast, caspase dependent apoptosis. Immuno-electron and confocal microscopy revealed affinity of both proteins to endocellular membranes. Both VP2 and VP3 were found to be bound to damaged ER and mitochondrial membranes. Interaction of the proteins with membranes causing physical damage of organelles...
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Gene Therapy of CML: Experimental Vaccines against Bcr-abl-transformed Cells
Lučanský, Vincent ; Vonka, Vladimír (advisor) ; Roubalová, Kateřina (referee) ; Reiniš, Milan (referee)
Chronic myeloid leukemia is malignant disease characterized by myeloproliferative clonal expansion of hematopoietic stem cell. It is causally associated with the formation of the so called Philadelphia chromosome and production of its specific product, the chimeric BCR-ABL protein. The amino acid sequence of the fusion region is unique, implying that the BCR-ABL protein carries tumor specific antigen. Currently imatinib mesylate dominates the treatment of CML. It is well tolerated and when compared to the other drugs used, it prolongs the life expectancy significantly. Unfortunately, it is not capable to cure the disease. The only potentially curative approach nowadays is the bone marrow transplantation; however, it is connected with a relatively high morbidity and mortality. Moreover, it is available only to a minority of the patients. Under these circumstances the need for the development of a relatively safe and generally available treatment is understandable. Immunotherapy could be such a treatment. Several experimental vaccines based on BCR-ABL sequence were developed and tested in mice in our institute. The DNA vaccines used were carrying sequences coding for the whole BCR-ABL protein, or for 25 amino acids long junction region (these DNA sequences were fused with adjuvant genes such as...
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"Features of the tumor microenvironment and their utilisation for drug delivery"
Větvička, David ; Hovorka, Ondřej (advisor) ; Reiniš, Milan (referee) ; Drbal, Karel (referee)
- David Větvička, M.Sc . Many researchers have, in the past, focused on pathophysiological features of tumor tissue, various tumor-nonmalignant cell interactions, and secretion of active molecules within the tumor mass. All these aspects of tumor structure are known as tumor microenvironment. The composition of particular tumor ecosystem is highly variable, with differences between various tumor types, even between patients with the same diagnosis, and in separate areas of the same tumor. Moreover, further changes in tumor microenvironment often occur during the progression of the disease. Studies of tumor microenvironment have revealed both novel targets for therapy and new prognostic markers. New therapy modalities are being developed to target these discovered features, including drugs functioning to boost anti-malignancy immunity, to block pro-metastatic potential, or to utilize the unique features of this pathological environment established by the tumor. These are obviously of great interest and harbor high potential for better management of malignant diseases. The focus of this thesis is to study the interactions of polymeric drug delivery systems within the tumor microenvironment and to utilize various features of this specific niche for drug delivery research. We have followed three...
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Epigenetic mechanisms in the regulation of the B7-H1 and IRF-1 expression in tumour cells.
Hrušková, Veronika ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
Interferon γ is an important T-cell helper type 1 (Th1) cytokine involves in antimicrobial immunity. It is a part of the inflammatory immune response in the site of infection. However, for its proper function, the regulation of immunity is necessary to avoid injury of the tissue caused by long-term inflammation. While interferon γ triggers expression of proinflammatory genes, it also regulates genes which inactivate immune response. The B7-H1 molecule belongs among these inhibitory regulators. Furthermore, antitumour effect of interferon γ is well-known as well. After extensive experiments, interferon γ was tested as an immunotherapeutic drug against melanomas in clinical trials. However, the trials had to be terminated prematurely because of unsuccessful results. It started to be clear that interferon γ could have also a protumour effect. Interferon γ upregulates the expression of B7-H1 molecule which aids tumour in escape from immunity. The B7-H1 molecule possesses a binding site for interferon regulatory factor 1 (IRF-1) in its promoter region. This IRF-1 is induced by interferon γ - JAK/STAT signalling pathway. In our previous research, we observed interferon γ induced DNA demethylation of promoters in genes that are involved in antigen presenting machinery. Additionally, DNA methylation of...
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STAT3 signalling pathway modulation and its impacts on cell phenotype, senescence and secretome.
Novotný, Ondřej ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
Cellular senescence represents an effective barrier to tumour growth on the one hand, on the other hand, senescent cells secrete substances that create a protumourigenic environment. Understanding and influencing these processes will bring new approaches in the fight against tumours and other serious diseases. The IL-6/JAK2/STAT3 signalling pathway is crucial both in oncogenesis and in the induction of cellular senescence and it also has a major influence on the composition of the secretome of senescent cells. Targeted blockade of this pathway could therefore change the tumour microenvironment, either by directly affecting the induction of senescence itself, or by adjusting secretion and thereby by attenuation of the immunosuppressive environment induced by produced cytokines. The aim of this thesis is to find out whether and how inhibitors of the STAT3 signalling pathway can change the secretome of senescent and proliferating tumour cells. Based on the comparison of the toxicity and effectiveness of the STAT3 signalling pathway inhibition by Stattic analogues on proliferating TC-1 tumour cells, I selected two suitable inhibitor molecules for further experiments. I found out that these selected inhibitors significantly reduce the secretion of important pro-tumour signalling molecules by TC-1 tumour...
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Combined immunotherapy of tumors with different expression of MHC class I molecules
Piataková, Adrianna Julia ; Šmahel, Michal (advisor) ; Krulová, Magdaléna (referee) ; Reiniš, Milan (referee)
Immunotherapy experienced ups and downs before being recognized as a paramount therapy for cancer. Evidence from the latest studies revealed that the tumour microenvironment (TME) plays a decisive role in the outcome of immunotherapeutic treatment. In addition, one of the mechanisms used by cancer cells to evade immunosurveillance is reduction of the expression of major histocompatibility complex class I (MHC-I), by which cancer cells become invisible to cytotoxic T lymphocytes (CTLs). Therefore, cancer immunotherapy should involve combined strategies to target both tumour cells and TME from different sites by activating other immune cells in addition to CTLs, such as tumour-associated macrophages (TAMs). This Ph.D. thesis aimed to investigate combined immunotherapy, composed of DNA immunization, immunostimulatory compounds, and an immune checkpoint inhibitor to activate adaptive and innate immunity and inhibit immunosuppression, respectively. For this purpose, murine models related to HPV-16-induced tumours with either reversibly (TC-1/A9 cell line) or irreversibly (TC-1/dB2m) reduced MHC-I expression were used. The development of the TC-1/dB2m clone was a part of this project and this clone was obtained by deactivating the B2m gene. An important focus of the research was the analysis of TAMs isolated from...
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Vliv chemoterapie a genotoxického stresu na imunologické vlastnosti nádorových buněk.
Horňáková, Michaela ; Reiniš, Milan (advisor) ; Drbal, Karel (referee)
Cancer treatment includes the use of chemotherapeutic agents, which have various effects on tumour cells, such as direct toxicity to cancer cells, immunogenic cell death induction and changes in cancer cells phenotype. Throughout the last decade many researchers have been focusing on the induction of genotoxic stress and cellular senescence, which chemotherapy can trigger. Even though induction of senescence in cancer cells represents an important mechanism for tumour suppression, there has been increasing evidence that shifting cancer cells into a senescent state by chemotherapy is not always beneficial. Senescent cells are associated with a specific secretory phenotype, which allows such cells to alter their microenvironment, modulate anti-tumour immunity, induce tumour suppression and even promote cancer development. Therefore, senescent cells elimination by innate or specific immunity, which can be boosted by immunotherapy, can be an important barrier preventing tumour growth. Powered by TCPDF (www.tcpdf.org)
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Interaction between NKT and myeloid derived suppressor cells and antitumour immunity
Straňavová, Lucia ; Reiniš, Milan (advisor) ; Holáň, Vladimír (referee)
Myeloid- derived suppressor cells (MDSCs) are a heterogeneous population of cells, which plays an important role in the suppression of anti-tumor immune responses. NKT cells represent an additional heterogeneous cell population that plays a crucial role in the regulation of immune responses. It shows that MDSCs and NKT cells may be similar to other populations imunoregulatory cells interact with each other and influence their functions. These interactions are important regulatory factor that may contribute to activation and to suppress anti-tumor immunity. Through interactions with type I NKT cells could differentiate these immunosuppressive MDSCs to immunogenic APC, which could form the basis for immunotherapeutic vaccine. All interactions between the NKT and MDSCs but have a positive effect of imunoregulatory. Interaction between MDSCs and CD4 + NKT cells II. type are immunosuppressive and may subsequently suppress the activity of cytotoxic T-lymphocyte (CTL). In some tumor models it was found that the immunosuppressive nature may also be interactions between MDSCs and type I NKT cells He had, however, alleviate the use of all-trans-retinol acid (ATRA), which induces differentiation of MDSCs.
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Induction of the immune response against HPV16-associated tumours with experimental vaccines
Kalenská, Romana ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
5 Induction of the immune response against HPV16-associated tumours with experimental vaccines The E6/E7 oncoproteins of human papillomaviruses are expressed in most trans- formed cells of cervical carcinoma and, therefore, are attractive targets for T cell-mediated immunotherapy. We have investigated the capacity of vaccines based on E7 oncoprotein- derived peptides to induce cellular immune responses and their therapeutic potential for treatment of minimal residual disease after surgery in a murine experimental model mi- micking human HPV16-associated carcinomas. We compared the effect of E749-57 peptide (RAHYNIVTF) exhibiting immunodominant epitope recognized by cytotoxic T lymphocy- tes with E744-62 peptide (QAEPDRAHYNIVTFCCKCD = 8Q) exhibiting CTL, TH and B- cell epitopes. Immune responses were compared in healthy mice and in mice after surgery or chemotherapy of tumours with ifosfamide derivative CBM-4A. Cellular immune re- sponses were monitored in spleen cells of C57BL/6 mice using ELISPOT-IFN-γ and 51 Cr- release assay. Flow cytometry was used for the detection of CD4+ , CD8+ , CD4+ CD25+ , Gr-1+ CD11b+ and CD3+ NK1.1+ populations. Vaccination with 8Q peptide and synthetic CpG oligodeoxynucleotide as adjuvant induced stronger antitumour immune responses than immunodominant CTL epitope alone in both...
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