The Darkness of the God according Gregory of Nyssa

Diplomová práce s názvem Temnota Boží podle Řehoře z Nyssy si klade za cíl zjistit, co považuje Řehoř za temnotu Boží a jak ji interpretuje na základě biblického příběhu o Mojžíši. Primárním zdrojem pro interpretaci temnoty je Řehořova kniha Život Mojžíšův. Kniha Život Mojžíšův čtenáři představuje jednotlivé fáze Mojžíšova hledání Boha z hlediska smyslu literárního i kontemplativního. Sekundárním zdrojem objasňujícím Řehořovo učení a výklad temnoty je kniha Lenky Karfíkové s názvem Řehoř z Nyssy: Boží a lidská nekonečnost. Diplomová práce je rozdělená do šesti hlavních kapitol. V prvních třech kapitolách se čtenář dozví kontext, do kterého Řehoř vstupuje, a který ho ovlivňuje v rámci teologické tradice. Také Řehořovo teologické učení a důležité pojmy s ním spojené. Kapitola čtyři až šest slouží k přemostění teoretické nauky a k praktické aplikaci Řehořova učení na duchovní život člověka. Čtenář se dozví, co to znamená v duchovním životě spatřit Boha v temnotě. Diplomová práce představuje temnotu Boží jako způsob přemýšlení o Bohu, vrchol duchovní cesty člověka, i jako jednu z Božích teofanií (zjevení). Klíčová slova: Řehoř z Nyssy, Mojžíšův život, Boží Trojice, temnota Boží, duchovní cestaSummary: The diploma thesis is called The Darkness of the God acording to Gregory of Nyssa. The aim of this diploma thesis is to find out what Gregory of Nyssa means by term Darkness of the God and how he interprets it on basis of biblical story of Moses. Primary information source for interpretation of the Darkness is Gregory's book The Life of Moses. The book presents particular phases of Moseses looking for God from literary nad contemplative point of view. The book Gregory of Nyssa: God's and human intinity from Lenka Karlíková is secondary information source which explaining Gregory's teaching and interpretation of the Darkness. The diploma thesis is devided to six main chapters. First three deal with kontext which Gregory entries and which infuences him in terms of theological tradition, as well as Gregory's theological teaching and related important terms. Chapters four to six connect theoretical teaching and practical application within human spiritual life. Reader finds out in practice, what it means within the spiritual life to see God in the Darkness. The diploma thesis interprets Darkness of the God as a way of thinking of God, the top of human spiritual journey and as one of God's theophanies. Keywords: Gregory of Nyssa, The Life of Moses, The Holy Trinity, The Darkness of the God, the...Ecumenical InstituteEkumenický institutProtestant Theological FacultyEvangelická teologická fakult

Hudba jako efektivní metodický prostředek ve výuce anglické výslovosti

Záměrem této diplomové práce je poukázat na prospěšný účinek hudby při výuce anglické výslovnosti. Teoretická část popisuje, jak hudba pozitivně ovlivňuje proces učení a také pomáhá vytvářet efektivní prostředí pro vyučování. Práce se dále zaměřuje na motivující vliv hudby a na její schopnost zvýšit zájem studentů při výuce. Praktická část seznamuje čtenáře s konkrétními příklady metod používaných v hodinách zpěvu na základní umělecké škole se zaměřením na vybrané rysy anglické výslovnosti. Klíčová slova: hudba, výslovnost, zpěv, píseňThe aim of this thesis is to point out the beneficial effect music has on learning English pronunciation. The theoretical part describes how music can positively influence the process of learning and helps to create an effective educational environment. This work further focuses on the motivational effect of music and on its ability to increase student interest. The practical part provides the reader with concrete examples of the methodology used in singing classes at the basic art schools with focus on selected features of English pronunciation. Key words: music, pronunciation, singing, songKatedra anglického jazyka a literaturyFaculty of EducationPedagogická fakult

Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation

Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2′, which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications

Computational and Structural Evidence for Neurotransmitter-mediated Modulation of the Oligomeric States of Human Insulin in Storage Granules

Human insulin is a pivotal protein hormone controlling metabolism, growth and ageing, and whose malfunctioning underlies diabetes, some cancers and neuro-degeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available, which fall into three conformational states: T6, T3Rf3 and R6. As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic β-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations

Characterization of Viral Insulins Reveals White Adipose Tissue Specific Effects in Mice [preprint]

Members of the insulin/IGF superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single chain (sc), i.e. IGF-1-like, forms of the viral insulin/IGF-1 like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e. more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1). GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A, IR-B) and to the IGF1R, and for the latter show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments in awake mice revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75nmol/kg/min) stimulated a comparable glucose uptake in heart, skeletal muscle and brown adipose tissue, GIV dcVILP stimulated ~2 fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin. Taken together, these results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogues that specifically target the tissues, and provide new insights into their potential role in disease

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain : Chemical Synthesis and Biological Activities

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications

Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains

Characterization of insulin crystalline form in isolated β-cell secretory granules

Insulin is stored in vivo inside the pancreatic β-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic β-cell insulin secretory granules should promote insulin crystalline state in the form of Zn2+-stabilized hexamers. Electron microscopic images of thin sections of the pancreatic β-cells often show a dense, regular pattern core, suggesting the presence of insulin crystals. However, the structural features of the storage forms of insulin in native preparations of secretory granules are unknown, because of their small size, fragile character and difficult handling. We isolated and investigated the secretory granules from MIN6 cells under near-native conditions, using cryo-electron microscopic (Cryo-EM) techniques. The analysis of these data from multiple intra-granular crystals revealed two different rhomboidal crystal lattices. The minor lattice has unit cell parameters (a ≃ b ≃ 84.0 Å, c ≃ 35.2 Å), similar to in vitro crystallized human 4Zn2+-insulin hexamer, whereas the largely prevalent unit cell has more than double c-axis (a ≃ b ≃ c ≃ 96.5 Å) that probably corresponds to two or three insulin hexamers in the asymmetric unit. Our experimental data show that insulin can be present in pancreatic MIN6 cell granules in a microcrystalline form, probably consisting of 4Zn2+-hexamers of this hormone

Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone : Receptor Binding Specificity

Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain