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2-DE analysis of hepatocyte secretome under hyperhomocysteinemia
Chrudinová, Martina ; Ryšlavá, Helena (advisor) ; Novák, Petr (referee)
Homocysteine is a plasmatic, sulphur-containing amino acid derived from methionin. It is an amino acid not used in protein synthesis and it's role is to serve as an intermediate in numerous metabolic pathaways. Elevated concentrations of homocysteine in blood are associated with many pathologies, as cardiovascular and neurodegenerative diseases, diabetes or osteoporosis, thus homocysteine has been a common subject of many biochemical experiments in last two decades. For this bachelor thesis, the proteomic comparison of proteins secreted by primary human hepatocytes under 2mM homocysteine and primary human hepatocytes with no homocysteine added to the incubation medium was made, using large-gel two-dimensional electrophoresis. Hepatocytes were incubated in serum free medium for 48 hours. Proteins secreted to medium were precipitated by acetonitrile. Two dimensional electrophoresis was made. Protein maps were analysed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (Bio-Rad, USA). Search criteria for differentially secreted proteins were three-fold change at the signicance level 95 %, Student's t-test. These proteins were cut off the preparative gels and identified by tandem mass spectrometry. In total, there were 17 spots equal to 15 proteins found as differentially exprimed proteins. Among these...
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Immune response in mammalian species against progenitor cell types including induced pluripotent stem cells (iPSCs)
Kovandová, Barbora ; Drbal, Karel (advisor) ; Krulová, Magdaléna (referee)
Stem cells may be very useful tool for regenerative medicine. They are able to repair any tissue in a human body and cure any damage caused by injury, sickness or aging. But at first, we have to deal with problems, which are connected with their usage - especially their immunogenicity. This bachelor thesis is focused on immunogenicity of embryonal (ESC), induced pluripotent (iPSC) and adult stem cells (ASC). Tissues derived from ESC are in vivo described as strongly immunogenic, although they seem to be immunosuppressive in vitro. Another danger of their usage is their tumorigenic potential. There also exist ethical issues connected with their usage. iPSC were supposed to be a good replacement for ESC, because no immunological nor ethical problems were expected. Surprisingly, they were described as immunogenic, too, even in autologous environment. These cells were also described as tumorigenic; this is the main reason for now why they cannot be used for the replacement therapy. Immunogenicity, so as tumorigenicity of iPSC may be a consequence of their dedifferentiation from somatic back to stem cells. ASC are the only stem cells, which are already used for the replacement therapy (transplantation of bone marrow). Some of them are described as immunosuppressive or tumor-suppressive, other are...
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Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants
Kollárová, Nikola ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...
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Effect of Wharton's jelly-derived mesenchymal stem cells secretome in the treatment of rat spinal cord injury
Jančová, Pavlína ; Chudíčková, Milada (advisor) ; Grobárová, Valéria (referee)
As previously shown, transplantation of Wharton's jelly-derived mesenchymal stem cells in the experimental model of spinal cord injury leads to the motor and sensory functions improvement, supports neuroregeneration, angiogenesis and provides immunomodulation. On the other hand, these cells have limited migration and survival capacity, and their therapeutic effect is mediated mostly by their secretome. Therefore, application of mesenchymal stem cells derived conditioned medium is studied as an alternative option for cell therapy. In this thesis, therapeutic effect of repeated intrathecal delivery of human Wharton's jelly-derived mesenchymal stem cells and their conditioned media in the treatment of spinal cord injury was compared. After induction of a balloon ischemic compression lesion, stem cells or conditioned media were administered weekly in three doses. Behavioral analyses were carried out up to nine weeks after spinal cord injury and revealed significant improvement of both treated groups compared to the untreated saline control. Application of stem cells and conditioned media also resulted in a higher amount of spared tissue and enhanced expression of genes related to neuroregeneration, although the size of glial scar was not reduced. Compared to application of stem cells, application of...
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Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants
Kollárová, Nikola ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...
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Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants
Kollárová, Nikola ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...
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Immune response in mammalian species against progenitor cell types including induced pluripotent stem cells (iPSCs)
Kovandová, Barbora ; Drbal, Karel (advisor) ; Krulová, Magdaléna (referee)
Stem cells may be very useful tool for regenerative medicine. They are able to repair any tissue in a human body and cure any damage caused by injury, sickness or aging. But at first, we have to deal with problems, which are connected with their usage - especially their immunogenicity. This bachelor thesis is focused on immunogenicity of embryonal (ESC), induced pluripotent (iPSC) and adult stem cells (ASC). Tissues derived from ESC are in vivo described as strongly immunogenic, although they seem to be immunosuppressive in vitro. Another danger of their usage is their tumorigenic potential. There also exist ethical issues connected with their usage. iPSC were supposed to be a good replacement for ESC, because no immunological nor ethical problems were expected. Surprisingly, they were described as immunogenic, too, even in autologous environment. These cells were also described as tumorigenic; this is the main reason for now why they cannot be used for the replacement therapy. Immunogenicity, so as tumorigenicity of iPSC may be a consequence of their dedifferentiation from somatic back to stem cells. ASC are the only stem cells, which are already used for the replacement therapy (transplantation of bone marrow). Some of them are described as immunosuppressive or tumor-suppressive, other are...
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2-DE analysis of hepatocyte secretome under hyperhomocysteinemia
Chrudinová, Martina ; Ryšlavá, Helena (advisor) ; Novák, Petr (referee)
Homocysteine is a plasmatic, sulphur-containing amino acid derived from methionin. It is an amino acid not used in protein synthesis and it's role is to serve as an intermediate in numerous metabolic pathaways. Elevated concentrations of homocysteine in blood are associated with many pathologies, as cardiovascular and neurodegenerative diseases, diabetes or osteoporosis, thus homocysteine has been a common subject of many biochemical experiments in last two decades. For this bachelor thesis, the proteomic comparison of proteins secreted by primary human hepatocytes under 2mM homocysteine and primary human hepatocytes with no homocysteine added to the incubation medium was made, using large-gel two-dimensional electrophoresis. Hepatocytes were incubated in serum free medium for 48 hours. Proteins secreted to medium were precipitated by acetonitrile. Two dimensional electrophoresis was made. Protein maps were analysed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (Bio-Rad, USA). Search criteria for differentially secreted proteins were three-fold change at the signicance level 95 %, Student's t-test. These proteins were cut off the preparative gels and identified by tandem mass spectrometry. In total, there were 17 spots equal to 15 proteins found as differentially exprimed proteins. Among these...
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Proteomics of biological fluids
Jarkovská, Karla ; Kovářová, Hana (advisor) ; Petrák, Jiří (referee) ; Ulčová-Gallová, Zdeňka (referee)
Reproductive diseases, mainly those resulting in the infertility affect the chances of human being to reproduce. On the contrary, the heart disease, cancer and degenerative diseases currently account for majority of deaths in the world. Usually, these lifestyle diseases need longer lifespan to become the cause of death. The proteins secreted by cells carry important information about the cell's well-being, as well as about the condition of the tissues formed by these cells. Once secreted, these proteins may also be transferred throughout the body by means of body fluids, many of which are easily accessible for further 'in-depth' studies. Cellular and secreted proteins are often a focus of studies using proteomic means and the revelation of protein alterations can lead us to new ideas about the molecular mechanisms of diseases as well as possible identification of proteins that may be used as new targets for pharmaceutical intervention or molecules that could be used for diagnostic or prognostic purposes. Taking into consideration the above aspects, this research was undertaken to find proteins that could: (a) characterise the human follicular fluid as microenvironment of the maturing oocyte, to increase understanding of reproductive processes to improve the techniques of assisted repro- duction;...
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