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P53 protein isoforms production and purification in the bacterial expression system
Vadovičová, Natália ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.
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Analysis of TP53 gene transcriptional varians in human leucemic cells
Vlahová, Veronika ; Mgr.Diana Grochová, Ph.D. (referee) ; Malčíková,, Jitka (advisor)
The theoretical part of the thesis summarizes general information about the human TP53 gene and p53 protein, which is encoded by this gene. There is also dealt with the transcriptional variants - p53 isoforms. The experimental part is focused on the detection of isoform p53 and its shorter, yet undescribed form. The starting material is peripheral blood of patients from University Hospital Brno, from which RNA was isolated. Subsequently, RNA was transcribed by reverse transcription into cDNA which was amplified by polymerase chain reaction (PCR). Experimental measurements demonstrate that p53 occurs in all the B-lymphocytes of patients with chronic lymphocytic leukemia. It also demonstrats the existence of a shorter form of p53.
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P53 protein isoforms production and purification in the bacterial expression system
Vadovičová, Natália ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.
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The role of estrogen receptors in prognosis and therapy outcome of breast cancer
Kloudová, Alžběta ; Souček, Pavel (advisor) ; Dračínská, Helena (referee)
Estrogen receptors (ER) are members of nuclear receptor family, which mediate distinct physiological functions after binding a steroid ligand. Apart from that they play a role in many diseases including breast cancer. ER is among proteins routinely evaluated in clinical practice and on the basis of ER expression, patients are treated by endocrine therapy. There are different opinions of the role of ER in cancer cells, but in the future, detection of ER and treatment by ER- and ER-selective ligands could contribute to improvement of cancer therapy. Isoforms, mutations and posttranslational modifications of ER present other important factors, which can influence estrogen signalization and endocrine therapy efficiency and deciphering of their importance for cancer cells could bring better understanding of ER signalization and improvement of the therapy.
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Impact of weight loss in obese subjects on the sensitivity of adipose tissue cells in relation to stress of endoplasmatic reticulum.
Karlická, Michaela ; Rossmeislová, Lenka (advisor) ; Němcová, Vlasta (referee)
Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In obesity their production is dysregulated, which is one of the major factors contributing to the onset of a chronic low-grade systemic inflammation representing a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis was to analyze the secretion of selected adipocytokines (adiponectin, IL6 and MCP1) by in-vitro differentiated adipocytes, isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. In case of adiponectin, the secretion of its isoforms was analyzed too. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. At the same time the development of the endoplasmic reticulum (ER) in the course of adipogenesis was monitored by indirect...
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The role of WT1 and its isoforms in normal haematopoiesis and leukaemogenesis
Kramarzová, Karolina ; Trka, Jan (advisor) ; Pospíšilová, Dagmar (referee) ; Živný, Jan (referee)
61 Summary Wilms' tumor gene 1 (WT1) is highly expressed in acute leukemia and other hematological malignancies. It has been therefore suggested as a potential universal marker of minimal residual disease (MRD), particularly in patients with acute myeloid leukemia (AML). Due to controversial results of some of the studies, the role of WT1 in MRD follow-up and WT1 prognostic significance remain unclear. WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, despite the increasing number of studies, the clinical impact of WT1 and its isoforms in acute leukemia have not yet been elucidated. We established a unique qPCR method to assess the expression pattern of the main 4 WT1 isoforms. Using this method, we determined the ratio of WT1 variants in the samples of patients with AML, myelodysplastic syndrome (MDS) and healthy controls. Our data showed that this pattern can distinguish among particular hematological malignancies, but lacks a prognostic significance. Within our international study group we determined the prognostic significance of total WT1 expression in childhood AML. Based on our results of a large cohort of patients we can conclude that WT1 expression at...
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Analysis of TP53 gene transcriptional varians in human leucemic cells
Vlahová, Veronika ; Mgr.Diana Grochová, Ph.D. (referee) ; Malčíková,, Jitka (advisor)
The theoretical part of the thesis summarizes general information about the human TP53 gene and p53 protein, which is encoded by this gene. There is also dealt with the transcriptional variants - p53 isoforms. The experimental part is focused on the detection of isoform p53 and its shorter, yet undescribed form. The starting material is peripheral blood of patients from University Hospital Brno, from which RNA was isolated. Subsequently, RNA was transcribed by reverse transcription into cDNA which was amplified by polymerase chain reaction (PCR). Experimental measurements demonstrate that p53 occurs in all the B-lymphocytes of patients with chronic lymphocytic leukemia. It also demonstrats the existence of a shorter form of p53.
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