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Monitoring of bone metabolism affected by selected drugs
Gradošová, Iveta ; Živná, Helena (advisor) ; Broulík, Petr (referee) ; Blahoš, Jaroslav (referee)
Monitoring of bone metabolism affected by selected drugs Osteoporosis is one of the most common metabolic bone diseases, which belong to civilization diseases, and is a major health and socioeconomic problem, particularly in the older age groups. Cardiovascular diseases are one of the great problems of our society and a leading cause of death worldwide. The major risk factors include hypercholesterolemia and arterial hypertension, which can be effectively reduced by several groups of drugs. At the present, not much attention has been paid to whether or how these drugs affect bone metabolism. With increasing age, people are more likely to develop hypertension and hypercholesterolemia with progressive loss of bone leading to osteoporosis. Many studies have suggested that antihypertensive and hypolipidemic drugs in some way influence bone metabolism. The subject of the present thesis was to investigate the effect of selected, frequently prescribed antihypertensive drugs (amlodipine, metoprolol), and hypolipidemic drugs (ezetimibe, atorvastatin) on bone metabolism in healthy male Wistar albino rats and in rats after orchidectomy (Wistar and spontaneously hypertensive rats). During my postgradual study, three experiments in rats with above mentioned drugs were performed. In the first experiment, drugs...
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Studies on molecular interactions of the mu-opioid and TRPV1 receptors
Melkes, Barbora ; Novotný, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Krůšek, Jan (referee)
In this work, we investigated the behavior of the -opioid receptor (MOR) and the transient receptor potential vanilloid 1 (TRPV1) ion channel in the plasma membrane and their mutual communication. Both these receptors are implicated in pain perception and analgesia. We observed that the lateral mobility of MOR was strongly affected by different biased opioid agonists. DAMGO and endomorphin-2 display opposite bias towards MOR. According to our results, they also have the opposite effects on the mobility of MOR. Morphine induced only small changes in the mobility of MOR. Moreover, cholesterol depletion and blockage of G protein signaling by pertussis toxin (PTX) affected the ability of different MOR agonists to alter MOR mobility in a unique manner. The effects of DAMGO and endomorphin-2 were compromised under these conditions. On the other hand, we observed increased movement of MOR after the addition of morphine. PTX alone did not affect receptor movement, but it completely disrupted the effect of cholesterol depletion on morphine induced changes the mobility of MOR. Next we studied the mobility of TRPV1. The TRPV1 agonist capsaicin changed the lateral mobility of TRPV1. Surprisingly, after adding the MOR antagonist naloxone, the apparent diffusion coefficient of TRPV1 but to a lower extent than...
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Influence of protein SGIP1 on partners participating in signalization of cannabinoid receptor 1
Pejšková, Lucie ; Blahoš, Jaroslav (advisor) ; Novotný, Jiří (referee)
The G-protein-coupled receptor (GPCR) family represents the largest family of cell surface receptors. GPCRs are activated by endogenous or exogenous ligands, and are targets for more than a quarter of currently used drugs. Activation of receptors initiates intracellular signaling pathways. This way the membrane receptors transfer information from the outside environment into the cell. Based on the signal the cell can respond to the changes of the environment. Key observation important for this thesis is interplay of cannabinoid and opioid signaling in vivo, which can have significant physiological effects1 . Cannabinoid receptor 1 (CB1R) and µ opioid receptor (MOR) belong to the rhodopsin family of receptors, and both are coupled with Gαi/o proteins2 . Both are located in certain areas in central nervous system (CNS) and share a lot of important features. Activation of both of the receptors leads to inhibition of adenylyl cyclase, thus decreasing the level of cyclic adenosine monophosphate in the cell, and modulates extracellular regulated kinase 1 and 2 (ERK1/2)2 . In view of the numerous anatomical, biochemical and pharmacological evidence supporting the existence of the functional interaction between opioid3 and cannabinoid receptor systems this topic became interesting for our research. In our...
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Structure and Function of Glutamate Carboxypeptidase II
Šácha, Pavel ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Blahoš, Jaroslav (referee)
4 Závěr GCPII je důležitý protein, který hraje roli v mnoha fyziologických i patologických procesech. Proto bylo třeba získat větší množství enzymaticky aktivního proteinu pro jeho další biochemický výzkum. Heterologní expresí v hmyzích buňkách S2 bylo exprimováno a následně purifikováno dostatečné množství velmi čistého a aktivního enzymu. To umožnilo jeho biochemickou charakterizaci, krystalizaci a později vedlo i k vyřešení krystalové struktury. GCPII je aktivní v širokém rozmezí pH 6 - 8, s maximem kolem pH 7,5. Zjistili jsme, že kromě přirozeného substrátu Ac-Asp-Glu GCPII štěpí také acetylované dipeptidy Ac-Asp-Met, Ac-Glu-Met, Ac-Glu-Glu, Ac-Ala-Glu a Ac-Ala-Met. U těchto substrátů byly změřeny kinetické parametry štěpení. Nalezení dalších substrátů může vést k objevení dosud nepopsaných fyziologických rolí GCPII. Také byly porovnány hodnoty IC50 inhibitorů známých z literatury u námi připravené GCPII a GCPII izolované z potkaních mozků. IC50 bylo u všech méně specifických inhibitorů nižší u rekombinantní GCPII než u GCPII izolované z mozků. Rozdíly ve výsledcích vysvětlujeme vazbou méně specifických inhibitorů na jiné proteiny preparátů z mozku. Vůbec nepochybujeme o tom, že data získaná za použití čistého rekombinantního proteinu jsou přesnější než ta, ktará byla získána ze špatně definovaných...
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Structure, activity and metabolism of human glutamate carboxypeptidase II
Mlčochová, Petra ; Konvalinka, Jan (advisor) ; Blahoš, Jaroslav (referee) ; Šedo, Aleksi (referee)
CONCLUSIONS AND PERSPECTIVES Recentry reported crystal sructures of GCpII provide stucturar insight into the organization of the substrate binding cavity and highlight residues implicated in substrate / inhibitor binding in the sI site of the enzyme. To comprement and extend the s[ucturar studies, we constructed a QMzMM model of GCPII in complex with its substrate, N-aceýt. aspartyl-glutamate, which enabled us to pÍedict additional anrino acid residues interacting with the bound subsrate. and used site-directed mutagenesis to assess the contribution of individual residues for substrate ,/ inhibitor binding and enzymatic activity of GCpII. we prepared and characterized 12 GcpJ' mutants targeting the amino acids in the vicinity of substrate./inhibitor binding pockets. The experimental results suggest that residues (especiaily Arg210) in the sť site are critical for substrate./inhibitor binding, whereas the residues forming the sl pocket niight be more important for the .fine-tuning, of GCptr substrate specificity and appear to be relevant for substrate turnover and may play a role in the enzyme's mechanism of action. Even though the QIVýMM calculations of the NAAG binding mode in the GCPII active site enabled us to predict the structure and enzyme-substrate interactions in the sl binding site, the complete...
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Role of proteins associated with the Cannabinoid receptor 1 in endocannabinoid signaling
Vozárová, Denisa ; Blahoš, Jaroslav (advisor) ; Novotný, Jiří (referee)
To preserve homeostasis and proper function in every living organism, it is important for cells to communicate with each other and their environment. Cells are constantly processing a huge amount of extracellular stimuli through proteins called receptors. Receptors can transduce the signal from extracellular to intracellular compartments. G- protein coupled receptors are the biggest group, in which also belongs Cannabinoid receptor type 1 (CB1R). Endocannabinoid system regulates many biological processes such as learning, food intake, and movement. Obesity is a serious issue nowadays and in cases of claryfing its molecular-genetic basis, there was found Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 has a role in the regulation of energetic balance and its overexpression is leading to a development of obesity. SGIP1 was detected as an interaction partner of CB1R and it had been found that it is involved in internalization via clathrin-mediated endocytosis (CME). Key proteins for initiation and early phase of CME are FCHO1/2, with which SGIP1 shares high sequential homology. However, effect of SGIP1 on internalization of activated CB1R is inhibitory unlike FCHO1/2,wheras detailed mechanism of its function remains unclear. The aim of this...
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Role of proteins associated with the cannabinoid receptor 1 in endocannabinoid signaling
Vozárová, Denisa ; Blahoš, Jaroslav (advisor) ; Novotný, Jiří (referee)
To preserve homeostasis and proper function in every living organism, it is important for cells to communicate with each other and their environment. Cells are constantly processing a huge amount of extracellular stimuli through proteins called receptors. Receptors can transduce the signal from extracellular to intracellular compartments. G-protein coupled receptors are the biggest group, in which also belongs Cannabinoid receptor type 1 (CB1R). Endocannabinoid system regulates many biological processes such as learning, food intake, and movement. Obesity is a serious issue nowadays and in cases of searching for candidate molecules, there was found Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 has a role in the regulation of energetic balance and its overexpression is leading to a development of obesity. SGIP1 was detected as an interaction partner of CB1R and it had been found that it is involved in internalization via clathrin-mediated endocytosis. SGIP1 is very homological with FCHO1/2 - important proteins which participate on early stages of endocytosis. Mechanism of inhibitory effect of SGIP1 on internalization remains unclear. The aim of this study is to clarify the role of distinct domains of SGIP1 in context of endocytosis. Key...
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The role of protein SGIP1 in regulation of Cannabinoid Receptor 1
Chlupisová, Lenka ; Blahoš, Jaroslav (advisor) ; Novotný, Jiří (referee)
Mutual cell communication in the human body ensures the proper functioning of the essential mechanisms necessary for the life of the individual and preserving the homeostasis of the whole organism. Such communication is established by various types of signal transmission from the recipient cell to the donor cell, depending on the location and type of communicating cells. One such type is signalization through receptor molecules found on the surface or within the cell receiving the signal. These receptors receive the signal molecule in the form of a ligand and bind it to themselves, while activating the receptor and then triggering the intracellular signaling pathways. The most widely represented receptors in the eukaryotic organism include G-protein-coupled receptors, which represent signaling ensured by activation of the intracellular G-protein complex, and one of the main mechanisms occurring in neuronal signaling and signal transmission in the form of a neurotransmitter. Regulation of the amount of receptors on the surface of the cell and transport of the signal molecule into the intracellular spaces of the cell is ensured by the mechanism of endocytosis, whereby internalization of the ligand- bound receptor in the cytoplasm occurs. One of the most researched mechanisms is clatharin-mediated...
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Characterization of molecular components in cannabinoid signaling pathways.
Hájková, Alena ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Maletínská, Lenka (referee)
The cannabinoid receptor 1 (CB1R), a member of the G-protein coupled receptors superfamily, is a key player in endocannabinoid signalling. The CB1R is found presynaptically in neurones where it modulates synaptic plasticity. Precise description of the molecular mechanisms of synaptic neurotransmission is crucial for understanding of brain diseases and development of new therapeutic aproaches. Possible pharmacological targets of CB1R signalling include the treatment of various ailments such as energy imbalance disorders (anorexia, obesity), drug addiction, pain, insomnia, and some psychiatric conditions. This study reveals the "Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1" (SGIP1) as a novel interacting partner of the CB1R. The SGIP1 is an intracellular neuronal protein localized predominantly in axon terminals and is involved in clathrin mediated endocytosis. The overexpression of SGIP1 imbalance energy homeostasis and leads to obesity. We show that SGIP1 affects CB1R signalling via ERK1/2 whereas G-protein signallization remains unaltered. The SGIP1 also hinders CB1R internalization from the cell surface and supports its interaction with β-arrestin2. Also, we demonstrated heterodimerization of the main splice variants of metabotropic glutamate...
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Bone remodeling in rheumatic diseases: Bone loss in juvenile idiopathic arthritis
Brábníková Marešová, Kristýna ; Štěpán, Jan (advisor) ; Blahoš, Jaroslav (referee) ; Hrnčíř, Zbyněk (referee)
Introduction: The inflammation plays the essential role in the bone loss in juvenile idiopathic arthritis (JIA). Proinflammatory cytokines and also glucocorticoids (GCs) may activate bone resorption by osteoclasts. Simultaneously, bone formation can be attenuated, especially by inhibitors of proteins, which control the osteoblast differentiation. The aim was to verify the hypothesis that in patients with highly active JIA, reduction of bone formation via Wingless (Wnt) proteins inhibitors - Dickkopf 1 (Dkk-1) and sclerostin could be found. Except the densitometry measurements of bone and lean mass, we assessed markers of disease activity, bone metabolism and remodeling in young adult patients with JIA before and during 2 years of anti TNFα (tumour necrosis factor α) treatment, which decreases disease activity. Results: In patients with JIA before antiTNFα treatment, bone mineral density (BMD, g/cmš) was significantly reduced compared to controls. Values of BMD and body composition in JIA significantly depended on disease duration and GCs treatment. Serum concentration of sclerostin was significantly elevated in JIA compared to values in healthy controls. Values of the other monitored markers did not differ between JIA and controls. In patients with JIA, Dkk-1 correlated positively with C-reactive...
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