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Effects of allopregnanolon on motor functions of young laboratory rat.
Krejčí, Lukáš ; Mareš, Pavel (advisor) ; Valeš, Karel (referee)
Allopregnanolone is a typical representative of neurosteroids. It can be formed by de novo synthesis directly in the CNS or as a metabolite of progesterone. It is an allosteric modulator of GABAA receptors, due to which it has anxiolytic, sedative and anticonvulsant effects. This work investigates the effect of allopregnanolone (ALLO) on the locomotor skills of laboratory rat pups. The results will serve as a reference study for the work of Tereza Košťálová (2020), who investigated the newly discovered neuroactive steroid pregnanolone pyroglutamate (PPG). This exogenous steroid, synthesized at the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic (IOCB PRAGUE), offers potential use in the treatment of epilepsy. Our goal was to perform the same battery of motor tests after the application of allopregnanolone as a comparison, to what extent and in what way these two substances acutely affect the motor performance of the rat pup. The theoretical part of the thesis describes the physiological mechanisms of neurosteroids in the human body and their potential in the treatment of serious diseases, especially epilepsy. Next, the ontogenetic development of the rat's motor skills is described and compared to humans. The methodology describes the selection and...
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Identification of new neuroactive steroids that are able to interact with allosteric binding sites on purinergic P2X receptors
Sivčev, Sonja ; Zemková, Hana (advisor) ; Vyklický, Vojtěch (referee) ; Stojilkovic, Stanko S. (referee)
(EN) Purinergic P2X receptors are ATP-gated cation channels with multiple physiological roles and are emerging as important therapeutic targets in a range of diseases. P2X subunit consists of two transmembrane helices (TM1 and TM2), an extracellular ATP-binding domain, and intracellular N- and C- termini. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. P2X are ubiquitously expressed. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain. The activity of P2X depends not only on the presence of ATP but also on allosteric modulators that may inhibit or potentiate the activity of these channels. Our aim was to identify new molecules that could interact with allosteric binding sites on P2X receptors, design and synthesize new analogues of neurosteroids, and define crucial receptor domains and amino acids important for neurosteroid binding. By using a patch-clamp electrophysiology technique we recorded ATP-induced currents in HEK293T cells transfected with rat P2X2, P2X4, and P2X7, as well as in the rat anterior pituitary cells and hypothalamic neurons endogenously expressing these receptors. We found that 17β-ester derivatives of testosterone, namely testosterone butyrate and...
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Neurosteroid modulation of ligand-gated ion-channel activity
Krausová, Barbora ; Vyklický, Ladislav (advisor) ; Moravec, Jan (referee)
The term neurosteroids refers to steroids that are synthetized in the nervous tissue from cholesterol or steroidal precursors from peripheral sources. These compounds affect the neuronal excitability by modulating the function of some ligand-gated ion channels. NMDA (N methyl D aspartate) receptors are glutamate gated ion channels involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. GABAA ( aminobutyric acid type A) receptors mediate most of the inhibitory synaptic transmission in the mammalian brain and are targeted by many clinically important drugs. Function of NMDA and GABAA receptors can by affected by neurosteroids, both positively and negatively. The aim of this work is to summarize the current knowledge about the neurosteroid effects on the function of GABAA a NMDA receptors and suggest the physiological role and the potential therapeutic use of the neurosteroids as a regulator of some functions of the central nervous system.
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Neurosteroid effects on intracellular calcium and excitotoxicity
Naimová, Žaneta ; Smejkalová, Terézia (advisor) ; Adámek, Pavel (referee)
NMDA receptors belong to the family of ionotropic glutamate receptors, and are involved in synaptic plasticity, learning and memory. However, overactivation by the agonist glutamate can lead to neuronal death - excitotoxicity. Exitotoxicity is a result of excessive calcium influx into the cell through NMDA receptors, and is associated with many cental nervous system (CNS) diseases. Neurosteroids are endogenous compounds capable of NMDA receptor modulation, thus they may have pharmacological potential in the treatment of CNS disorders. The aim of this work was to investigate how pregnanolone sulfate (PA-S) and pregnanolone hemipimelate (PA-hPim) influence somatic calcium and excitotoxicity. We used fluorescence microscopy for recording changes in somatic calcium concentration. We observed that PA-S had no influence on relative somatic calcium concentration. Synthetic analog PA-hPim increased somatic calcium levels slightly. Next, we used oxygen-glucose deprivation (OGD) in vitro to study the influence of neurosteroids on excitotoxicity. Both PA-S and PA-hPim were neuroprotective in the model of acute OGD in vitro. Moreover, PA-S or PA-hPim pretreatment induced ischemic tolerance to a subsequent OGD episode. Our results suggest that neurosteroids PA-S and PA-hPim are potential candidates for the development...
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Characterization of the effect of pregnanolone sulfate and its derivatives on NMDA receptors.
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...
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Ionotropic glutamate receptors and excitotoxicity
Skřenková, Kristýna ; Vyklický, Ladislav (advisor) ; Moravec, Jan (referee)
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and its excitatory role is mediated trough activation of glutamatergic ionotropic receptors which are responsible for synaptic transmission and play an important role in learning and memory formation. However, excessive exposure to glutamate can result in excitotoxicity which may lead to cell death. The following text is focused on one group of glutamate receptors - NMDA receptors. The study of the receptors is in the centre of current neurobiology research because there is a series of experimental and clinical evidences that they directly participate in the development of serious diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and may cause neuronal damage in trauma, hypoxia and embolia. The aim of this bachelor thesis is to give a brief overview of current knowledge about the structure and function of NMDA receptors and mechanisms of their activation which leads to excitotoxicity and related neuroprotection.
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Effects of sulfated neurosteroids on proteins involved in excitatory synaptic transmission
Naimová, Žaneta ; Smejkalová, Terézia (advisor) ; Mrózková, Petra (referee)
The discovery of steroid compounds capable of synthesize or accumulation in CNS and PNS led to a question about their function. Neurosteroid compounds are capable of modulating synaptic transmission. Effect is direct and fast mediated through nongenomic mechanisms. They are known to affect wide array of channels and receptors - both excitatory and inhibitory. This thesis summarizes findings about effect of sulfated neurosteroid on proteins involved in excitatory synaptic transmission. Thesis covers findings about ionotropic glutamate receptors, TRP channels, metabotropic receptors, sodium and potassium channels. Excessive or insufficient activity of these proteins involved in synaptic transmission can lead to a pathological condition. The purpose of this thesis is to summarize findings about effect of these compounds, point out structural and function characteristic probably responsible for their action and to outline possible pharmacological usage.
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Synthesis and Properties of Neuroactive Steroids
Kapras, Vojtěch
Herein is reported the synthesis of molecular probes for action of neuroactive steroids in vitro and in living organisms. In the first part, preparation of enantiomeric pregnane steroids is investigated, ultimately resulting into the total synthesis of ent-progesterone. The chirality of the target molecule is introduced by a highly effective organocatalytic asymmetric Robinson annulation. A new method for the sequential construction of five-membered carbocyclic ring is introduced as the key step. This is composed of substrate-controlled copper-catalyzed conjugate addition followed by radical oxygenation and subsequent thermal cyclization employing the persistent radical effect. The synthesis of truncated neurosteroid analogs is described and their biological activity at the NMDA receptor is compared with the native hormone. In the second part, methodology for specific deuterium labeling of both angular methyls of the 5β-pregnane steroid core is explored. Special attention was paid to the Barton-McCombie deoxygenation as the tool for introduction of the last deuterium atom into the methyl group. Both positions were labelled with total of three deuterium atoms in high isotopic purity.
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Neurosteroid effects on intracellular calcium and excitotoxicity
Naimová, Žaneta ; Smejkalová, Terézia (advisor) ; Adámek, Pavel (referee)
NMDA receptors belong to the family of ionotropic glutamate receptors, and are involved in synaptic plasticity, learning and memory. However, overactivation by the agonist glutamate can lead to neuronal death - excitotoxicity. Exitotoxicity is a result of excessive calcium influx into the cell through NMDA receptors, and is associated with many cental nervous system (CNS) diseases. Neurosteroids are endogenous compounds capable of NMDA receptor modulation, thus they may have pharmacological potential in the treatment of CNS disorders. The aim of this work was to investigate how pregnanolone sulfate (PA-S) and pregnanolone hemipimelate (PA-hPim) influence somatic calcium and excitotoxicity. We used fluorescence microscopy for recording changes in somatic calcium concentration. We observed that PA-S had no influence on relative somatic calcium concentration. Synthetic analog PA-hPim increased somatic calcium levels slightly. Next, we used oxygen-glucose deprivation (OGD) in vitro to study the influence of neurosteroids on excitotoxicity. Both PA-S and PA-hPim were neuroprotective in the model of acute OGD in vitro. Moreover, PA-S or PA-hPim pretreatment induced ischemic tolerance to a subsequent OGD episode. Our results suggest that neurosteroids PA-S and PA-hPim are potential candidates for the development...
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Effects of sulfated neurosteroids on proteins involved in excitatory synaptic transmission
Naimová, Žaneta ; Smejkalová, Terézia (advisor) ; Mrózková, Petra (referee)
The discovery of steroid compounds capable of synthesize or accumulation in CNS and PNS led to a question about their function. Neurosteroid compounds are capable of modulating synaptic transmission. Effect is direct and fast mediated through nongenomic mechanisms. They are known to affect wide array of channels and receptors - both excitatory and inhibitory. This thesis summarizes findings about effect of sulfated neurosteroid on proteins involved in excitatory synaptic transmission. Thesis covers findings about ionotropic glutamate receptors, TRP channels, metabotropic receptors, sodium and potassium channels. Excessive or insufficient activity of these proteins involved in synaptic transmission can lead to a pathological condition. The purpose of this thesis is to summarize findings about effect of these compounds, point out structural and function characteristic probably responsible for their action and to outline possible pharmacological usage.
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