Among the peptidyl prolyl isomerases, the Cyclophilin family of proteins has been linked to various cellular activities such as regulation of homeostasis, mitochondrial permeability, and cell death. Their functionality spans throughout the cell and throughout all cell types as different isoforms. Previous studies done on Cyclophilin A revealed an interesting contact ensemble when bound to a substrate. Because of the similarity of CypA to its homologues, it is believed that they too will exhibit the same contact dynamics. We have defined the dynamics of cyclophilin isoforms through Molecular Dynamics simulations and determined their contact dynamics, characterizing their contact ensembles, and their relative dynamical conservation to each other.

Adaptation to chronic hypoxia is characterized by a variety of functional changes in order to maintain metabolic and energy homeostasis. It has been known for many years that both humans and animals indigenous or adapted to high-altitude hypoxia are more tolerant to an acute ischemic injury of the heart. HIF1α is found as a primary transcriptional regulator of adaptive response to hypoxia in all tissues, whereas HIF2α is more tissue specific. The activity of HIFα is regualted by prolyl hydoxylases (EGLN). The EGLN1 was shown to be more efficient in HIF1α hydroxylation than in HIF2α. As well as the EGLN3 is more specific for HIF2α . Under hypoxic conditions, HIF activates the transcription of about 70 genes. Hence, HIF1α and HIF2α can play an essential role in pathophysiology of ischemic heart disease. The aim of our study was to determine myocardial expression of HIFs and its regulatory hydroxylases in adult male Wistar rats adapted to continuous normobaric hypoxia (H; FIO2 = 0.1) for 3 weeks. Another two groups of rats were exposed to normobaric hypoxia intermittently for either 8 h/day (INH) or 23 h/day (RH) during the 3-week adaptation period. While H induces protective cardiac phenotype, the later regimen (RH) does not. The protein expression of HIFs, PHDs and FIH were then determined by...

Drabbová, A.: Title of Diploma Thesis: Centaurea cyanus L. (Asteraceae) alkaloids and their biological activity related to Alzheimer's disease. Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové 2020. Ethyl acetate and chloroform alkaloids extracts were prepared from Centaurea cyanus L. seeds. A novel alkaloid AD-1 (Adrabbin) was isolated from the ethyl acetate extract by common chromatografic methods (column chromatography, preparative TLC). Its structure was elucidated by mass spektrometry, NMR analysis and determination of optical rotation. The alkaloid AD-1 (Adrabbin) possesses a unique structure which consists of a cyclic indole moiety. Also LC-MS analysis of fractions obtained by flash chromatography was performed. In those fractions were detected molecular ions related to compounds previously isolated from other Centaurea species. Alkaloid AD-1 (Adrabbin) was tested on ability to inhibit human cholinesterases, prolyl oligopeptidase and gylcogen synthase kinase 3β. The compound was considered against human cholinesterases inactive (IC50 values > 100 µM). Interestibgly, the alkaloid inhibited prolyl oligopeptidase the same intensity as a standard berberine (AD-1: IC50 143,0 ± 6,0 µM; berberin: IC50 142,0 ± 21,0 µM). The novel compound...

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

V. Hojgrová: Alkaloids of Vinca minor L. and their biological activity VIII. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany. Number of pages 69. This diploma thesis deals with the isolation of alkaloids from Vinca minor L. from the family Apocynaceae. Separation of alkaloids from the selected fraction (VM 215-258) or from their subfractions (VM 34-41, VM 86, VM 87-113) was performed by preparative TLC. Two pure alkaloids were isolated from the subfraction (VH 34-41). The first VH-1 alkaloid not yet isolated and the second VH 2 alkaloid: (-)-raucubainin. Alkaloids were identified by EI-MS, LC-MS, NMR and optical rotation and were compared with data in the literature. Isolated alkaloids were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and prolyloligopeptidase (POP) inhibitory activity and for cytotoxicity. Both substances did not show significant cholinesterase inhibitory activity IC50 against AChE after measurement, only (-)-raucubainin showed a slight activity against BuChE (IC50 = 94 ± 7 μM), VH-1 was found to be inactive (IC50 > 100 μM). POP inhibitory activity has so far only been tested for (-)-raucubainin; was found to be inactive (IC50 > 1000 µM). The results of the cytotoxic activity of the alkaloids...

Celiac Disease (CD) is an autoimmune disorder that emerges due to the ingestion of gluten, a protein found in a variety of common grains such as wheat, rye, and barley. Approximately 1 in 100 individuals in the US suffer from CD, making it the most commonly diagnosed gastrointestinal disorder (Ciclitira et. al., 2005). These proline-rich gluten peptides are resistant to proteolysis and accumulate in the duodenum of the small intestine. Once in the duodenum, these peptides illicit an autoimmune response resulting in villous atrophy. Current treatment for CD requires a rigorous adherence to a gluten-free diet. Nevertheless, gluten-containing grains are ubiquitous in the western diet, so accidental exposure to gluten remains as a persistent threat. The approach of this project centers on genetically engineering a strain Lactobacillus reuteri to secrete a Myxococcus xanthus prolyl endopeptidase (PEP), an enzyme that hydrolyzes a peptide bond adjacent to an internal proline residue. The data from this study revealed that recombinant M. xanthus PEP purified from E. coli was effective in degrading Suc-Ala-Pro-pNA, a chromogenic substrate containing an internal proline residue. When introduced into a L. reuteri expression vector, mutations accumulated in the vector over the course of 5 days. These data suggested that toxicity was possibly associated with M. xanthus PEP and the amyl signal peptide.

The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in “physiological hypoxia” and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR.We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1/, Hif-p4h-2 hypomorph and Hif-p4h-3/ mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs.

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Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs would be most beneficial if administered soon after infarction, thus the aim of this project was to optimize CSC culture conditions to enhance their therapeutic potential for myocardial infarction. CSCs were isolated and expanded in vitro via the formation of cardiospheres to give cardiosphere-derived cells (CDCs). Neonatal rat CDCs were found to be heterogenous, containing cells expressing the cardiac stem cell marker, c-Kit, pluripotent cell markers, Oct-4, Sox 2, Klf-4 and Nanog, and early cardiac specific differentiation markers, Nkx 2.5 and GATA 4. Administration of CDCs to the infarcted rat heart increased the cardiac ejection fraction by 9%, capillary density by 9% and reduced scar volume by 33%, compared to the non-treated group. The proliferation rates and the expression of c-Kit were significantly decreased in CSCs isolated from aged rats and after extended culture in vitro, so, CSC culture was optimized using hypoxic preconditioning. Under hypoxia, CDC proliferation rates were 1.7-fold greater, and larger cardiosphere clusters were formed. Hypoxic CDCs had an increased cardiac stem cell population, in that c-Kit was increased by 220% and CD90 and CD105 were decreased by 55% and 35%, respectively, compared to normoxic CDCs. Further, hypoxia induced the expression of CXCR-4 (~3.2-fold), EPO (~3.0-fold) and VEGF (~1.5-fold), indicating that hypoxic preconditioning may stimulate stem cell homing and neovascularization in the infarcted myocardium. Notably, hypoxic CDCs were able to switch to anaerobic glycolytic metabolism and had approximately 80% lower oxygen consumption, suggesting that they may be better adapted to survive within the hypoxic infarct scar, compared with normoxic CDCs. Culture of CDCs with hypoxia-mimicking prolyl-4-hydroxylase inhibitors (PHDIs) using DMOG, BIC and a novel compound, EDBA, induced similar effects to hypoxic culture by increasing c-Kit, EPO, VEGF, CXCR-4, decreasing CD90 and CD105 and increasing glycolytic metabolism. However, PHDI treatment for 24 hours did not alter CDC proliferation rates and cells died after 24 hours. In conclusion, CDCs are a potential cell source for therapy after myocardial infarction and their therapeutic potential can be enhanced using hypoxia or PHDI-preconditioning techniques.

La flexibilité est une caractéristique intrinsèque des protéines qui doivent, dès le mo- ment de leur synthèse, passer d’un état de chaîne linéaire à un état de structure tridimen- sionnelle repliée et enzymatiquement active. Certaines protéines restent flexibles une fois repliées et subissent des changements de conformation de grande amplitude lors de leur cycle enzymatique. D’autres contiennent des segments si flexibles que leur structure ne peut être résolue par des méthodes expérimentales. Dans cette thèse, nous présentons notre application de méthodes in silico d’analyse de la flexibilité des protéines : • À l’aide des méthodes de dynamique moléculaire dirigée et d’échantillonnage pa- rapluie, nous avons caractérisé les trajectoires de liaison de l’inhibiteur Z-pro- prolinal à la protéine Prolyl oligopeptidase et identifié la trajectoire la plus pro- bable. Nos simulations ont aussi identifié un mode probable de recrutement des ligands utilisant une boucle flexible de 19 acides aminés à l’interface des deux domaines de la protéine. • En utilisant les méthodes de dynamique moléculaire traditionnelle et dirigée, nous avons examiné la stabilité de la protéine SAV1866 dans sa forme fermée insérée dans une membrane lipidique et étudié un des modes d’ouverture possibles par la séparation de ses domaines liant le nucléotide. • Nous avons adapté auproblème de la prédiction de la structure des longues boucles flexibles la méthode d’activation et de relaxation ART-nouveau précédemment uti- lisée dans l’étude du repliement et de l’agrégation de protéines. Appliqué au replie- ment de boucles de 8 à 20 acides aminés, la méthode démontre une dépendance quadratique du temps d’exécution sur la longueur des boucles, rendant possible l’étude de boucles encore plus longues. / Flexibility is an intrinsic characteristic of proteins who from the moment of synthesis into a linear chain of amino acids, have to adopt an enzymatically active tridimensionnel structure. Some proteins stay flexible once folded and display large amplitude confor- mational changes during their enzymatic cycles. Others contain parts that are so flexible that their structure can’t be resolved using experimental methods. In this thesis, we present our application of in silico methods to the study of protein flexibility. • Using steered molecular dynamics and umbrella sampling, we characterized the binding trajectories of the Z-pro-prolinal inhibiter to the Prolyl oligopeptidase pro- tein and we identified the most probable trajectory. Our simulations also found a possible ligand recrutement mechanism that involves a 19 amino acids flexible loop at the interface of the two domains of the protein. • Using traditional and steered molecular dynamics, we examined the stability of the SAV1866 protein in its closed conformation in a lipid membrane and we studied one of its proposed opening modes by separating its nucleotide binding domains. • We also adapted the activation-relaxation technique ART-nouveau which was pre- viously used to study protein folding and aggregation to the problem of structure prediction of large flexible loops. When tested on loops of 8 to 20 amino acids, the method demonstrate a quadratic execution time dependance on the loop length, which makes it possible to use the method on even larger loops.