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Proteomic architecture of sperm-egg interactions
Otčenášková, Tereza ; Stopka, Pavel (advisor) ; Petr, Jaroslav (referee) ; Vrbacký, Marek (referee)
Recent advances in proteomic methods provide new insights for biological research including the field of reproductive biology. Determination of the proteomic basis of spermatozoa is pivotal for understanding the complex process of gamete interactions during fertilization such as acrosome reaction. Great differences imposed by postcopulatory sexual selection and phylogeny can be observed regarding the size, shape, and molecular composition of sperm across animal taxa. The first objective of this doctoral thesis is to characterize the protein contents of the acrosome to ascertain its further functional significance in sperm-egg interaction. Also, we aim to investigate the potential relationships between sperm protein composition and sperm morphology diversification, risk of sperm competition, and species phylogenetic background. Wild-caught males from natural populations of species of Mus musculus musculus, Apodemus flavicollis, Microtus arvalis (order Rodentia), Acrocephalus palustris, Chloris chloris, Phylloscopus collybita, Cinclus cinclus, Hirundo rustica, and Taeniopygia guttata from a captive population (order Passeriformes) were subject to the analyses. Nano-liquid chromatography with tandem mass spectrometry was applied as the main methodological approach in this thesis. Our data implicate...
Proteomics for beginners
Hubálek, Martin ; Vrbacký, M. ; Křenková, Alena
Proteomic tutorial for the beginners will summarise and present current methods to analyse and evaluate proteomic samples. The tutorial will be divided into two parts. The first part will focus on data accquisition process and interpretation of spectra. The second part will focus on protein quantification.
Functional characterisation of new components of mitochondrial proteome.
Kovalčíková, Jana ; Vrbacký, Marek (advisor) ; Červinková, Zuzana (referee) ; Ješina, Pavel (referee)
1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...
Expression of the recombinant extracellular parts of human leukocyte receptors LLT1 and NKR-P1A
Vostárek, František ; Novák, Petr (advisor) ; Vrbacký, Marek (referee)
NK cells are characterized as large granular lymphocytes that play important role in innate immunity. They are called as "first line defense", because of their capability to kill the target cells very fast, in a few minutes. They recognize the target cells using their surface receptors. This diploma thesis describes the preparation of extracellular domains of the human leukocyte receptor hNKR-P1A and its physiological ligand LLT1. The proteins were produced in E. coli as inclusion bodies, refolded in vitro by rapid dilution method (hNKR-P1A) and slow dilution method (LLT1). The proteins were purificated by chromatography and characterized by mass spectrometry techniques.
The role of NADPH oxidase and ros in invadopodia formation
Hanušová, Kristýna ; Brábek, Jan (advisor) ; Vrbacký, Marek (referee)
Invadopodia as specific organelles enabling tumour cells movement, spreading over the organism and ultimately formation of metastasis are possible and promising targets of tumour therapy. Recently, many interesting facts about assembly and mechanism of function of invadopodia were discovered. Invadopodia are centres of ECM degradation by extra-cellular proteases facilitating an invasion of tumour cells. For creation of invadopodia a precisely localized increased production of ROS is necessary. ROS work as crucial signalling molecules and participate in many processes resulting in invadopodia formation. ROS in tumour cells are produced by specific extra-mitochondrial NADPH oxidases (Nox). Several regulatory molecules participating in activation and localization of Nox to invadopodia have been discovered recently (Tks organizer proteins). Furthermore, a regulatory role of Src kinase in ROS production and subsequent invadopodia formation was confirmed. Key words: ECM degradation, invadopodia, invasion, proteases, Nox, ROS, Src kinase, Tks proteins
The Role of Tyrosine Kinase Activity of Mitochondrial ERBB2/HER2 in Breast Cancer
Novotná, Eliška ; Rohlena, Jakub (advisor) ; Vrbacký, Marek (referee)
Breast cancer is a common malignant disease affecting millions of women worldwide. Amplification of HER2 oncogene, a tyrosine kinase receptor, in breast cancer allows application of targeted therapy, but approximately one third of patients develop resistance to treatment. Relocalization of HER2 from the plasma membrane into the mitochondria was found and suggested as one of the potential causes of such resistance. Here we document that the function of mitochondrial HER2 is distinct from that of HER2 in the plasma membrane. Mitochondrial HER2 enhances cancer cell energetic metabolism, proliferation and migration in vitro, and tumour formation in vivo in mice correlating with elevated level of ROS signalling. The kinase activity of mitochondrial HER2 is unaffected, therefore I investigated its role in mitochondrial HER2 function. Moderate, endogenous levels of the kinase activity of mitochondrial HER2 drive pro-tumorigenic properties of breast cancer cells, while constitutive kinase activity sensitizes these cells to cell death and attenuates tumour formation in animal models. On the other hand, impairment of kinase activity due to mutation in the ATP binding site of mitochondrial HER2 supports adherence-independent growth in vitro and tumor growth in vivo. We propose that HER2 function in...
The Role of Tyrosine Kinase Activity of Mitochondrial ERBB2/HER2 in Breast Cancer
Novotná, Eliška ; Rohlena, Jakub (advisor) ; Vrbacký, Marek (referee)
Breast cancer is a common malignant disease affecting millions of women worldwide. Amplification of HER2 oncogene, a tyrosine kinase receptor, in breast cancer allows application of targeted therapy, but approximately one third of patients develop resistance to treatment. Relocalization of HER2 from the plasma membrane into the mitochondria was found and suggested as one of the potential causes of such resistance. Here we document that the function of mitochondrial HER2 is distinct from that of HER2 in the plasma membrane. Mitochondrial HER2 enhances cancer cell energetic metabolism, proliferation and migration in vitro, and tumour formation in vivo in mice correlating with elevated level of ROS signalling. The kinase activity of mitochondrial HER2 is unaffected, therefore I investigated its role in mitochondrial HER2 function. Moderate, endogenous levels of the kinase activity of mitochondrial HER2 drive pro-tumorigenic properties of breast cancer cells, while constitutive kinase activity sensitizes these cells to cell death and attenuates tumour formation in animal models. On the other hand, impairment of kinase activity due to mutation in the ATP binding site of mitochondrial HER2 supports adherence-independent growth in vitro and tumor growth in vivo. We propose that HER2 function in...
Functional characterisation of new components of mitochondrial proteome.
Kovalčíková, Jana ; Vrbacký, Marek (advisor) ; Červinková, Zuzana (referee) ; Ješina, Pavel (referee)
1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...

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