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Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1
Dvořáková, Michaela ; Blahoš, Jaroslav (advisor) ; Konvalinka, Jan (referee) ; Stuchlík, Aleš (referee)
Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1 Abstract Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1) has been identified as an interacting partner of cannabinoid receptor 1 (CB1R). Their protein-protein interaction was confirmed by co-immunoprecipitation. SGIP1 hinders the internalization of activated CB1R and modulates its signaling in HEK293 cells. Employing whole-cell patch-clamp electrophysiology, we have shown that SGIP1 affects CB1R signaling in autaptic hippocampal neurons. Using a battery of behavioral tests in SGIP1 constitutive knock-out (SGIP1-/- ) and WT mice, we investigated the consequences of SGIP1 deletion on behavior regulated by the endocannabinoid system. In SGIP1-/- mice, exploratory levels, working memory and sensorimotor gating were unaltered. SGIP1-/- mice showed decreased anxiety-like and depressive-like behaviors. Fear extinction to tone was enhanced in SGIP1-/- females. Several cannabinoid tetrad behaviors were altered in the absence of SGIP1. SGIP1-/- males exhibited abnormal THC withdrawal behaviors. SGIP1 deletion also reduced acute nociception, and SGIP1-/- mice were more sensitive to antinociceptive effects of CB1R agonists and morphine. CB1R-SGIP1 interaction results in profound modification of CB1R...
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Role of NMDA NR1 subunit in pathophysiology of schizophrenia
Vrajová, Monika ; Horáček, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Vyklický, Ladislav (referee)
Our work is focused on the role of NR1 subunit of N-methyl-D-aspartate receptor in pathophysiology of schizophrenia. In animal model using separately or in combination, antisense oligodeoxynucleotide (aODN) for NR1, NR2A and NR2B subunit of NMDAR, we affected expression of these proteins in rat hippocampus. We assessed prepulse inhibition of acoustic startle reaction (PPI) in rats and protein expression of NMDAR subunits and expression of PSD proteins. There were significant differences in expression of PSD-95 and NR1 between groups. Application of aODN (NR2A, NR2B) was associated with a significant decrease of PSD-95. PPI and expression of NR2A, NR2B and PSD-93 were not changed after aODN application.The next part of the work concentrates on a human post mortem study. To assess actual changes in the expression of the NR1 subunit and its isoforms, we measured absolute differences in the levels of mRNA/protein for panNR1, as well as the individual mRNA/protein isoforms in the post mortem left/right hippocampus of patients with schizophrenia in comparison with non-psychiatric subjects. There were no significant differences in the panNR1 subunit mRNA expression, but the absolute left/right differences were much more pronounced in the patients with schizophrenia. The expression of splice variants in the...
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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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Neuropharmacology of spatial navigation, cognitive coordination and flexibility tests in animal models
Prokopová, Iva ; Stuchlík, Aleš (advisor) ; Vyklický, Ladislav (referee) ; Blahoš, Jaroslav (referee)
Spatial navigation, cognitive coordination and behavioral flexibility belong amongst cognitive functions, which play a role in many neuropsychiatric disorders. Behavioral tasks have proved to be useful paradigms to test these functions in pharmacological or genetic animal models. First aim was to determine a potential interaction between β-adrenergic and α1-adrenergic or D2-dopaminergic systems. Spatial navigation and coordination were impaired in both studies during co-aplication of subthreshold doses of drugs. Used substances belong to group of widely prescribed drugs, thus our results could be implicated in clinical practice. Another study examined an acute effect of MK-801 (animal model of schizophrenia) on behavioral flexibility in Carousel maze and the Morris water maze (MWM). Carousel maze showed higher sensitivity with impairments from 0.08 mg.kg-1 compared to 0.10 mg.kg- 1 in MWM. The final experiment aimed at testing the effect of reduced expression of Nogo-A protein on spatial navigation and behavioral flexibility of rats. A battery of tests in the Carousel maze revealed impairment in cognitive functions, MWM showed unaffected working memory of rats. Our results support the hypothesis linking Nogo-A knock-down rats with neuropsychiatric symptoms and cognitive disorders. Key words:...
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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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