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Protein profiling, metabolic enzymes and transmembrane signaling in the heart of spontaneously hypertensive SHR-Tg19 rat
Manakov, Dmitry ; Novotný, Jiří (advisor) ; Kuncová, Jitka (referee) ; Kalous, Martin (referee)
Cardiovascular diseases account for the majority of deaths both worldwide and in the Czech Republic. Main factors contributing heart disease development, aside age and sex, are obesity, high blood pressure and high blood cholesterol and triglyceride levels. Spontaneously hypertensive rat (SHR) was developed and used for search of genetic determinants of these traits. This commonly used rat model develops hypertension, dyslipidemia, and insulin resistance naturally which is caused by aberrant Cd36 fatty acid translocase gene. Previous studies have shown that rescue of Cd36 performed in the transgenic SHR-Tg19 strain enhances cardiac beta-adrenergic system, slightly increases heart mass and leads to higher susceptibility to arrhythmias. The present thesis had two main aims: 1) To investigate whether and how a transgenic rescue of Cd36 in SHR affects protein composition, mitochondrial function and activity of selected metabolic enzymes of the heart. 2) To study the expression and distribution of selected components of beta-adrenergic signaling system in lipid raft isolated form membranes using the TX-100 detergent. We set to compare two commonly used proteomic approaches, 2D electrophoresis with MALDI-TOF mass spectrometry and label-free LC-MS. The results did not reveal any overlap between...
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Protein profiling, metabolic enzymes and transmembrane signaling in the heart of spontaneously hypertensive SHR-Tg19 rat
Manakov, Dmitry ; Novotný, Jiří (advisor) ; Kuncová, Jitka (referee) ; Kalous, Martin (referee)
Cardiovascular diseases account for the majority of deaths both worldwide and in the Czech Republic. Main factors contributing heart disease development, aside age and sex, are obesity, high blood pressure and high blood cholesterol and triglyceride levels. Spontaneously hypertensive rat (SHR) was developed and used for search of genetic determinants of these traits. This commonly used rat model develops hypertension, dyslipidemia, and insulin resistance naturally which is caused by aberrant Cd36 fatty acid translocase gene. Previous studies have shown that rescue of Cd36 performed in the transgenic SHR-Tg19 strain enhances cardiac beta-adrenergic system, slightly increases heart mass and leads to higher susceptibility to arrhythmias. The present thesis had two main aims: 1) To investigate whether and how a transgenic rescue of Cd36 in SHR affects protein composition, mitochondrial function and activity of selected metabolic enzymes of the heart. 2) To study the expression and distribution of selected components of beta-adrenergic signaling system in lipid raft isolated form membranes using the TX-100 detergent. We set to compare two commonly used proteomic approaches, 2D electrophoresis with MALDI-TOF mass spectrometry and label-free LC-MS. The results did not reveal any overlap between...
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A study of beta-adrenergic myocardial signaling in spontaneously hypertensive rat of transgenic strain SHR-Tg19
Manakov, Dmitry ; Novotný, Jiří (advisor) ; Nováková, Olga (referee)
β-Adrenergic signalization plays an important role in heart, regulating cardiac frequency and contractility. It is also involved in development of hypertension and heart hypertrophy. Spontaneous hypertensive rat strain is a common model for human essential hypertension, although the origin of blood pressure abnormalities in SHR remains unknown. Dysfunction in the regulation of fatty acid translocase Cd36 was suggested as a link to development of hypertension in SHR. Transgenic strain SHR-Tg19 (also known as SHR-Cd36) was obtained, harboring a wild type of FAT/Cd36. This thesis aimed to investigate key elements of β-adrenergic signaling in the heart of SHR-Tg19 compared to their SHR controls. Expression and distribution of β1- and β2-ARs were measured using radioligand binding and Western blot analysis along with expression of selected G proteins and expression and activity of adenylyl cyclase. Our experiments showed that there were no significant changes in the Gsα and Giα subunits expressions, along with the amount of β1-AR in both left and right ventricles, according to the Western immunoblotting, but radioligand binding showed an increase in the quantity of β-ARs, particularly β2 subtype. Alongside, an increased expression of β2- ARs was observed in the right ventricle. Different...
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The role of FAT/CD36 trasporter in the pathophysiology of heart
Kovalčíková, Jana ; Nováková, Olga (advisor) ; Wasková, Petra (referee)
1. Abstract FAT/CD36 is an 88 kDa glycoprotein that plays a key role in the transport of long- chain fatty acids (LCFA) through the plasma membrane in heart, skeletal muscle and adipose tissue. It participates in the fatty acids (FA) transport together with other membrane proteins, which are fatty acid transport proteins (FATP1-6), the plasma membrane fatty acid binding protein (FABPpm) and cytosolic fatty acid binding protein (FABPc). In cardiac tissue are, except FAT/CD36, only represented FABPpm, FATP1 and 6 and heart type of FABPc, referred to as H-FABPc. In addition to this protein mediated FA transport, FA are already known to be transported by passive diffusion. The cell expression of FAT/CD36 is regulated by nuclear peroxisome proliferators- activated receptor (PPAR), in the heart primarily by PPAR- . Inactive FAT/CD36 is found in intracellular depots, while active FAT/CD36 is present on the plasma membrane in lipid rafts. The two most known pathways regulating the FAT/CD36 translocation from the depots to the membrane are the insulin signalling pathway, which involves the activation of the enzyme phosphatidyl-inositol-3-kinase (PI3K) and cardiac contraction activated cascade, which activates adenosinmonophosphate kinase (AMP kinase). Furthermore, FAT/CD36 can as well be regulated by ubiquitination...
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